Methods for Treating Lung Disorders

ABSTRACT

Disclosed herein are compositions and methods for treating lung disorders including lung tumors by pulmonary administration of compositions comprising taxane particles such as docetaxel or paclitaxel particles.

CROSS REFERENCE

This application claims priority to U.S. Provisional Patent ApplicationSer. Nos. 62/519,257 filed Jun. 14, 2017; 62/628,582 filed Feb. 9, 2018;62/653,942 filed Apr. 6, 2018; and 62/678,387 filed May 31, 2018, eachincorporated by reference herein in their entirety.

BACKGROUND

Lung cancer is the second most common cancer and one of the most lethal.Conventional therapies such as surgical resection, radiation, andchemotherapy have not resulted in satisfactory long-term survival rates.Systemic drug delivery, even at a high dose, results in only a limitedamount of taxane drugs reaching lung tumors. Improved methods fortreating lung tumors are thus needed.

SUMMARY OF THE INVENTION

In one aspect, the invention provides methods for treating a lungdisorder, such as a lung tumor or pulmonary fibrosis, comprisingpulmonary administration to a subject with a lung disorder of an amounteffective of a composition comprising taxane particles to treat the lungdisorder, wherein the taxane particles comprise at least 95% of thetaxane and have a mean particle size (number) of between 0.1 μm and 5μm. In one embodiment, the pulmonary administration may comprisenebulization, wherein the nebulizing results in pulmonary delivery tothe subject of aerosol droplets of the taxane particles or suspensionthereof. In another embodiment, the taxane particles may have a meanparticle size (number) of between 0.4 μm and 2 μm. In furtherembodiments, the taxane particles may have a mean particle size (number)of between about 0.4 μm and about 1.2 μm, or between about 0.6 μm andabout 1.0 μm.

In another embodiment, the taxane particles may have a specific surfacearea (SSA) of at least 10 m²/g, or at least 12 m²/g, 14 m²/g, 16 m²/g,18 m²/g, 20 m²/g, 25 m²/g, 30 m²/g, 32 m²/g, 34 m²/g, or 35 m²/g; orwherein the taxane particles have an SSA of between about 10 m²/g andabout 60 m²/g. In another embodiment, the taxane particles may bepresent in a suspension, wherein the suspension comprises:

(a) the taxane particles;

(b) a pharmaceutically acceptable carrier; and

(c) a polysorbate, wherein the polysorbate is present in the suspensionat a concentration of between about 0.01% v/v and about 1.5% v/v, orbetween about 0.01% v/v and about 1% v/v, about 0.01% v/v and about 0.5%v/v, about 0.01% v/v and about 0.4% v/v, about 0.01% v/v and about 0.25%v/v, about 0.05% v/v and about 0.5% v/v, about 0.05% v/v and about 0.25%v/v, about 0.1% v/v and about 0.5% v/v, about 0.1% v/v and about 0.25%v/v, about 0.1% v/v, about 0.16 v/v, or about 0.25% v/v. In oneembodiment, the pharmaceutically acceptable carrier may be saline, suchas 0.9% sodium chloride solution. In another embodiment, the polysorbatemay be polysorbate 80. In a further embodiment, the taxane may bepresent in the suspension at a concentration between about 1 mg/ml andabout 40 mg/ml, or about 6 mg/ml and about 20 mg/ml.

In another embodiment, the taxane particles and suspensions thereof maybe uncoated and exclude lipids, polymers, proteins such as albumin,polyethoxylated castor oil, and/or polyethylene glycol glyceridescomposed of mono-, di- and triglycerides and mono- and diesters ofpolyethylene glycol.

In other embodiments, the taxane may comprise paclitaxel, docetaxel,cabazitaxel, or a pharmaceutically acceptable salt thereof. In oneembodiment, the taxane may comprise paclitaxel or a pharmaceuticallyacceptable salt thereof; in this embodiment, the particles may have oneor more of the following characteristics:

(a) a mean bulk density (not tapped) between about 0.050 g/cm³ and about0.12 g/cm³, or between about 0.060 g/cm³ and about 0.11 g/cm³;

(b) a SSA of at least 12 m²/g, 15 m²/g, 18 m²/g, 20 m²/g, 25 m²/g, 30m²/g, 32 m²/g, 34 m²/g, or 35 m²/g;

(c) a SSA of between about 22 m²/g and about 40 m²/g, 25 m²/g and about40 m²/g, 30 m²/g and about 40 m²/g, or between about 35 m²/g and about40 m²/g; and/or

(d) wherein at least 40% (w/w) of the paclitaxel is dissolved in 30minutes or less in a solution of 50% methanol/50% water (v/v) at 37° C.and pH 7.0 in a USP II paddle apparatus operating at 75 RPM.

In another embodiment, the taxane particles may comprise docetaxel or apharmaceutically acceptable salt thereof, in this embodiment, theparticles may have one or more of the following characteristics:

(a) a mean bulk density (not tapped) between about 0.050 g/cm³ and about0.12 g/cm³, or between about 0.06 g/cm³ and about 0.1 g/cm³;

(b) a SSA of at least 12 m²/g, 15 m²/g, 18 m²/g, 20 m²/g, 25 m²/g, 30m²/g, 35 m²/g, 40 m²/g, or 42 m²/g;

(c) a SSA of between about 20 m²/g and about 50 m²/g, or between about35 m²/g and about 46 m²/g; and/or

(d) wherein at least 20% (w/w) of the docetaxel is dissolved in 30minutes or less in a solution of 15% methanol/85% water (v/v) at 37° C.and pH 7.0 in a USP II paddle apparatus operating at 75 RPM.

In another embodiment, the taxane particles may be in crystalline form.In a further embodiment, the taxane particles or suspensions thereof areaerosolized for administration, and the aerosol droplets have a massmedian aerodynamic diameter (MMAD) of between about 0.5 μm to about 6 μmdiameter, or between about 1 μm to about 3 μm diameter, or about 2 μm toabout 3 μm diameter.

In one embodiment, the taxane may remain detectable in lung tissue ofthe subject for at least 4 days after the administering.

In some embodiments, the taxane particles reside at the tumor site afteradministration of the composition exposing the tumor to the taxaneparticles for a sustained amount of time sufficient to stimulate theendogenous immune system of the subject resulting in the production oftumoricidal cells and infiltration of the tumoricidal cells into thetumor at a level sufficient to treat the tumor. In some embodiments, thestimulation of the endogenous immune systems produces a cellular(cell-mediated) immune response. In other embodiments, the stimulationof the endogenous immune system produces a humoral immune response. Insome embodiments, the stimulation of the endogenous immune systemproduces a tumor vaccine. In some embodiments, metastases are reduced oreliminated.

In one embodiment, the sustained amount of time is at least 4 weeks.

In some embodiments, the tumoricidal cells comprise T-cells, B cells, ornatural killer (NK) cells, or combinations thereof.

In some embodiments, the composition is administered in two or moreseparate administrations.

In some embodiments, the two or more separate administrations areadministered once a week for at least two weeks.

In other embodiments, the two or more separate administrations areadministered twice a week for at least one week, wherein the two or moreseparate administrations are separated by at least one day.

In some embodiments, the treatment of the tumor is elimination of thetumor.

Disclosed in the context of the present invention are the followingembodiments 1 to 25:

Embodiment 1 is a method for treating a lung disorder, including but notlimited to a lung tumor or pulmonary fibrosis, comprising pulmonaryadministration to a subject with a lung disorder of an amount effectiveof a composition comprising taxane particles to treat the lung disorder,wherein the taxane particles comprise at least 95% of the taxane andhave a mean particle size (number) of between 0.1 μm and 5 μm.

Embodiment 2 is the method of embodiment 1, wherein the pulmonaryadministration comprises nebulization, and wherein the nebulizingresults in pulmonary delivery to the subject of aerosol droplets of thetaxane particles or suspension thereof.

Embodiment 3 is the method of any one of embodiments 1-2, wherein thetaxane particles have a mean particle size (number) of between 0.4 μmand 2 μm.

Embodiment 4 is the method of any one of embodiments 1-3, wherein thetaxane particles have a mean particle size (number) of between about 0.4μm and about 1.2 μm, or between about 0.6 μm and about 1.0 μm.

Embodiment 5 is the method of any one of embodiments 1-4, wherein thetaxane particles have a specific surface area (SSA) of at least 10 m²/g,or at least 12 m²/g, 14 m²/g, 16 m²/g, 18 m²/g, 20 m²/g, 25 m²/g, 30m²/g, 32 m²/g, 34 m²/g, or 35 m²/g; or wherein the taxane particles havean SSA of between about 10 m²/g and about 60 m²/g.

Embodiment 6 is the method of any one of embodiments 1-5, wherein thetaxane particles are present in a suspension, wherein the suspensioncomprises:

-   -   (a) the taxane particles;    -   (b) a pharmaceutically acceptable carrier; and    -   (c) a polysorbate, wherein the polysorbate is present in the        suspension at a concentration of between about 0.01% v/v and        about 1.5% v/v, or between about 0.01% v/v and about 1% v/v,        about 0.01% v/v and about 0.5% v/v, about 0.01% v/v and about        0.4% v/v, about 0.01% v/v and about 0.25% v/v, about 0.05% v/v        and about 0.5% v/v, about 0.05% v/v and about 0.25% v/v, about        0.1% v/v and about 0.5% v/v, about 0.1% v/v and about 0.25% v/v,        about 0.1% v/v, about 0.16 v/v, or about 0.25% v/v.

Embodiment 7 is the method of embodiment 6, wherein the pharmaceuticallyacceptable carrier is saline, such as 0.9% sodium chloride solution.

Embodiment 8 is the method of embodiment 6 or 7, wherein the polysorbateis polysorbate 80.

Embodiment 9 is the method of any one of embodiments 6-8, wherein thetaxane is present in the suspension at a concentration between about 1mg/ml and about 40 mg/ml, or about 6 mg/ml and about 20 mg/ml.

Embodiment 10 is the method of any one of embodiments 1-9, wherein theparticles and suspensions thereof are uncoated and exclude lipids,polymers, proteins such as albumin, polyethoxylated castor oil, and/orpolyethylene glycol glycerides composed of mono-, di- and triglyceridesand mono- and diesters of polyethylene glycol.

Embodiment 11 is the method of any one of embodiments 1-10, wherein thetaxane comprises paclitaxel, docetaxel, cabazitaxel, or apharmaceutically acceptable salt thereof.

Embodiment 12 is the method of embodiment 11, wherein the taxanecomprises paclitaxel or a pharmaceutically acceptable salt thereof.

Embodiment 13 is the method of embodiment 12, wherein the particles haveone or more of the following characteristics:

-   -   (a) a mean bulk density (not tapped) between about 0.050 g/cm³        and about 0.12 g/cm³, or between about 0.060 g/cm³ and about        0.11 g/cm³;    -   (b) a SSA of at least 12 m²/g, 15 m²/g, 18 m²/g, 20 m²/g, 25        m²/g, 30 m²/g, 32 m²/g, 34 m²/g, or 35 m²/g;    -   (c) a SSA of between about 22 m²/g and about 40 m²/g, 25 m²/g        and about 40 m²/g, 30 m²/g and about 40 m²/g, or between about        35 m²/g and about 40 m²/g; and/or    -   (d) wherein at least 40% (w/w) of the paclitaxel is dissolved in        30 minutes or less in a solution of 50% methanol/50% water (v/v)        at 37° C. and pH 7.0 in a USP II paddle apparatus operating at        75 RPM.

Embodiment 14 is the method of embodiment 11, wherein the taxanecomprises docetaxel or a pharmaceutically acceptable salt thereof.

Embodiment 15 is the method of embodiment 14, wherein the particles haveone or more of the following characteristics:

-   -   (a) a mean bulk density (not tapped) between about 0.050 g/cm³        and about 0.12 g/cm³, or between about 0.06 g/cm³ and about 0.1        g/cm³;    -   (b) a SSA of at least 12 m²/g, 15 m²/g, 18 m²/g, 20 m²/g, 25        m²/g, 30 m²/g, 35 m²/g, 40 m²/g, or 42 m²/g;    -   (c) a SSA of between about 20 m²/g and about 50 m²/g, or between        about 35 m²/g and about 46 m²/g; and/or    -   (d) wherein at least 20% (w/w) of the docetaxel is dissolved in        30 minutes or less in a solution of 15% methanol/85% water (v/v)        at 37° C. and pH 7.0 in a USP II paddle apparatus operating at        75 RPM.

Embodiment 16 is the method of any one of embodiments 1-15, wherein thetaxane remains detectable in lung tissue of the subject for at least 4days after the administering.

Embodiment 17 is the method of any one of embodiments 1-16, wherein thetaxane particles are in crystalline form.

Embodiment 18 is the method of any one of embodiments 1-17, wherein thetaxane particles or suspensions thereof are aerosolized foradministration, and the aerosol droplets have a mass median aerodynamicdiameter (MMAD) of between about 0.5 μm to about 6 μm diameter, orbetween about 1 μm to about 3 μm diameter, or about 2 μm to about 3 μmdiameter. Embodiment 19 is the method of any one of embodiments 1-18,wherein the lung disorder comprises a lung tumor, and wherein the taxaneparticles reside at the tumor site after administration of thecomposition exposing the tumor to the taxane particles for a sustainedamount of time sufficient to stimulate the endogenous immune system ofthe subject resulting in the production of tumoricidal cells andinfiltration of the tumoricidal cells into the tumor at a levelsufficient to treat the tumor.

Embodiment 20 is the method of embodiment 19, wherein the sustainedamount of time is at least 4 weeks.

Embodiment 21 is the method of any one of embodiments 19-20, wherein thetumoricidal cells comprise T-cells, B cells, or natural killer (NK)cells, or combinations thereof.

Embodiment 22 is the method of any one of embodiments 1-21, wherein thecomposition is administered in two or more separate administrations.

Embodiment 23 is the method of embodiment 22, wherein the two or moreseparate administrations are administered once a week for at least twoweeks.

Embodiment 24 is the method of embodiment 22, wherein the two or moreseparate administrations are administered twice a week for at least oneweek, wherein the two or more separate administrations are separated byat least one day.

Embodiment 25 is the method of any one of embodiments 1-24, wherein thelung disorder comprises a lung tumor, and wherein the treatment of thetumor is elimination of the tumor.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph of lung tissue and plasma levels of paclitaxel overtime from inhalation study.

FIG. 2 is a plot of the aerodynamic diameter of a 6.0 mg/mL paclitaxelparticle formulation from inhalation study.

FIG. 3 is a plot of the aerodynamic diameter of a 20.0 mg/mL paclitaxelparticle formulation from inhalation study.

FIG. 4 is a graph of plasma levels of paclitaxel over time frominhalation study.

FIG. 5 is a graph of lung tissue levels of paclitaxel over time frominhalation study.

FIG. 6 is a graph of lung tissue and plasma levels of paclitaxel overtime from inhalation study.

FIG. 7 is a diagram of a compressed air jet Hospitak nebulizer.

FIG. 8 is a graph of animal body weight over time from Orthotopic LungCancer study.

FIG. 9 is a graph of animal body weight change over time from OrthotopicLung Cancer study.

FIG. 10 is a plot of animal lung weights from Orthotopic Lung Cancerstudy.

FIG. 11 is a plot of animal lung to body weight ratios from OrthotopicLung Cancer study.

FIG. 12 is a plot of animal lung to brain weight ratios from OrthotopicLung Cancer study.

FIG. 13 is a graph of average tumor areas from Orthotopic Lung Cancerstudy.

FIG. 14 is a plot of average tumor areas from Orthotopic Lung Cancerstudy.

FIG. 15 is a plot of tumor regression from Orthotopic Lung Cancer study.

FIG. 16 is a photomicrograph of Orthotopic Lung Cancer tissue slide—1006(Control) Adenocarcinoma-3, Primitive-1, Regression-0. Primarycharacteristics of the lung tumor masses. (2×).

FIG. 17 is a photomicrograph of Orthotopic Lung Cancer tissue slide—1006Control, Adenocarcinoma-3, Primitive-1, Regression-0. Primarycharacteristics of undifferentiated cells within the lung tumor masses.

FIG. 18 is a photomicrograph of Orthotopic Lung Cancer tissue slide—1006(Control) Adenocarcinoma-3, Primitive-1, Regression-0. Primarycharacteristics of undifferentiated cells within the lung tumor masses.

FIG. 19 is a photomicrograph of Orthotopic Lung Cancer tissue slide—1006(Control) Adenocarcinoma-3, Primitive-1, Regression-0. Primarycharacteristics of undifferentiated cells within the lung tumor massesshowing masses within alveolar spaces. a(20×).

FIG. 20 is a photomicrograph of Orthotopic Lung Cancer tissue slide—1006(Control) Adenocarcinoma-3, Primitive-1, Regression-0. Primarycharacteristics of primitive cells within the lung tumor masses. b(10×).

FIG. 21 is a photomicrograph of Orthotopic Lung Cancer tissue slide—1006(Control) Adenocarcinoma-3, Primitive-1, Regression-0. Primarycharacteristics of primitive cells within the lung tumor masses. b20×.

FIG. 22 is a photomicrograph of Orthotopic Lung Cancer tissue slide—1006(Control) Adenocarcinoma-3, Primitive-1, Regression-0. Primarycharacteristics of primitive cells within the lung tumor masses. b(40×).

FIG. 23 is a photomicrograph of Orthotopic Lung Cancer tissue slide—1006(Control) Adenocarcinoma-3, Primitive-1, Regression-0. Primarycharacteristics of primitive cells within the lung tumor masses. b(40×).

FIG. 24 is a photomicrograph of Orthotopic Lung Cancer tissue slide—1006(Control) Adenocarcinoma-3, Primitive-1, Regression-0 bronchiole.Primary characteristics of undifferentiated cells showing withinbronchiole. c(20×).

FIG. 25 is a photomicrograph of Orthotopic Lung Cancer tissue slide—1006(Control) Adenocarcinoma-3, Primitive-1, Regression-0 glands. Primarycharacteristics of acinar gland differentiation within the lung tumormasses. d(10×).

FIG. 26 is a photomicrograph of Orthotopic Lung Cancer tissue slide—1006(Control) Adenocarcinoma-3, Primitive-1, Regression-0 glands. Primarycharacteristics of acinar gland differentiation within the lung tumormasses. d(20×).

FIG. 27 is a photomicrograph of Orthotopic Lung Cancer tissue slide—2001(IV ABRAXANE®) Adenocarcinoma-2, Primitive-1, Regression-O. Primarycharacteristics of the lung tumor mass pushing underneath a bronchioleand showing no evidence of intravascular invasion. (2×).

FIG. 28 is a photomicrograph of Orthotopic Lung Cancer tissue slide—2001(IV ABRAXANE®) Adenocarcinoma-2, Primitive-1, Regression-0. Primarycharacteristics of the lung tumor mass pushing underneath a bronchioleand showing no evidence of intravascular invasion. (4×).

FIG. 29 is a photomicrograph of Orthotopic Lung Cancer tissue slide—2001(IV ABRAXANE®) Adenocarcinoma-2, Primitive-1, Regression-0. Primarycharacteristics of the lung tumor mass pushing underneath a bronchiole.(10×).

FIG. 30 is a photomicrograph of Orthotopic Lung Cancer tissue slide—2003(IV ABRAXANE®) Adenocarcinoma-1, Primitive-1, Regression-1.Characteristics of the lung tumor masses undergoing regression. (4×).

FIG. 31 is a photomicrograph of Orthotopic Lung Cancer tissue slide—2003(IV ABRAXANE®) Adenocarcinoma-1, Primitive-1, Regression-1.Characteristics of the lung tumor masses undergoing regression. (10×).

FIG. 32 is a photomicrograph of Orthotopic Lung Cancer tissue slide—2003(IV ABRAXANE®) Adenocarcinoma-1, Primitive-1, Regression-1.Characteristics of the lung tumor masses undergoing regression. (20×).

FIG. 33 is a photomicrograph of Orthotopic Lung Cancer tissue slide—2003(IV ABRAXANE®) Adenocarcinoma-1, Primitive-1, Regression-1.Characteristics of the lung tumor masses undergoing regression. Notelymphocytes and macrophages along the edge. 1(40×).

FIG. 34 is a photomicrograph of Orthotopic Lung Cancer tissue slide—2003(IV ABRAXANE®) Adenocarcinoma-1, Primitive-1, Regression-1.Characteristics of the lung tumor masses undergoing regression. Notelymphocytes and macrophages along the edge. 2(40×).

FIG. 35 is a photomicrograph of Orthotopic Lung Cancer tissue slide—2003(IV ABRAXANE®) Adenocarcinoma-1, Primitive-1, Regression-1.Characteristics of the lung tumor masses undergoing regression. Notelarger foamy and pigmented macrophages. 2, 2×(40×).

FIG. 36 is a photomicrograph of Orthotopic Lung Cancer tissue slide—2010(IV ABRAXANE®) Adenocarcinoma-3, Primitive-1, Regression-0. Primarycharacteristics of the lung tumor masses. (2×).

FIG. 37 is a photomicrograph of Orthotopic Lung Cancer tissue slide—2010(IV ABRAXANE®) Adenocarcinoma-3, Primitive-1, Regression-0. Primarycharacteristics of the lung tumor masses. (20×).

FIG. 38 is a photomicrograph of Orthotopic Lung Cancer tissue slide—2010(IV ABRAXANE®) Adenocarcinoma-3, Primitive-1, Regression-0. Primarycharacteristics of the lung tumor masses. Note subtle evidence ofmacrophages along the edge. (40×).

FIG. 39 is a photomicrograph of Orthotopic Lung Cancer tissue slide—4009(IH paclitaxel particle formulation 1× High) Adenocarcinoma-0,Primitive-0, Regression-4. Characteristics of the lung tumor masses thathave undergone complete regression. (2×).

FIG. 40 is a photomicrograph of Orthotopic Lung Cancer tissue slide—4009(IH paclitaxel particle formulation 1× High) Adenocarcinoma-0,Primitive-0, Regression-4. Characteristics of a lung tumor mass that hasundergone complete regression. Note stromal fibrosis. (10×).

FIG. 41 is a photomicrograph of Orthotopic Lung Cancer tissue slide—4009(IH paclitaxel particle formulation 1× High) Adenocarcinoma-0,Primitive-0, Regression-4. Characteristics of a lung tumor mass that hasundergone complete regression. Note stromal fibrosis, and lymphocytesand macrophages along the edge. (40×).

FIG. 42 is a photomicrograph of Orthotopic Lung Cancer tissue slide—5010(IH paclitaxel particle formulation 2× Low) Adenocarcinoma-1,Primitive-0, Regression-3. Characteristics of the lung tumor massesundergoing regression. (2×).

FIG. 43 is a photomicrograph of Orthotopic Lung Cancer tissue slide—5010(IH paclitaxel particle formulation 2× Low) Adenocarcinoma-1,Primitive-0, Regression-3. Characteristics a lung tumor mass that isundergoing regression. (10×).

FIG. 44 is a photomicrograph of Orthotopic Lung Cancer tissue slide—5010(IH paclitaxel particle formulation 2× Low) Adenocarcinoma-1,Primitive-0, Regression-3. Characteristics a lung tumor mass that isundergoing regression. (20×).

FIG. 45 is a photomicrograph of Orthotopic Lung Cancer tissue slide—5010(IH paclitaxel particle formulation 2× Low) Adenocarcinoma-1,Primitive-0, Regression-3. Characteristics a lung tumor mass that isundergoing regression. (40×).

FIG. 46 is a photomicrograph of Orthotopic Lung Cancer tissue slide—6005(IH paclitaxel particle formulation 2× High) Adenocarcinoma-1,Primitive-0, Regression-4. Characteristics a lung tumor mass that isundergoing regression. (2×).

FIG. 47 is a photomicrograph of Orthotopic Lung Cancer tissue slide—6005(IH paclitaxel particle formulation 2× High) Adenocarcinoma-1,Primitive-0, Regression-4. Characteristics a lung tumor mass that isundergoing regression. Note stromal fibrosis, and lymphocytes andmacrophages along the edge. (10×).

FIG. 48 is a photomicrograph of Orthotopic Lung Cancer tissue slide—6005(IH paclitaxel particle formulation 2× High) Adenocarcinoma-1,Primitive-0, Regression-4. Characteristics a lung tumor mass that isundergoing regression. Note lymphocytes and macrophages along the edge.(20×).

FIG. 49 is a photomicrograph of Orthotopic Lung Cancer tissue slide—6005(IH paclitaxel particle formulation 2× High) Adenocarcinoma-1,Primitive-0, Regression-4. Note lymphocytes and macrophages along theedge. (40×).

FIG. 50 is a photomicrograph of Orthotopic Lung Cancer tissue slide—6005(IH paclitaxel particle formulation 2× High) Adenocarcinoma-1,Primitive-0, Regression-4. Note the presence of a focal area of residualtumor cells within an alveolus. 2(40×).

FIG. 51 is a graph of animal body weight over time from inhalationstudy.

FIG. 52 is a graph of animal body weight change over time frominhalation study.

FIG. 53 is a graph of plasma levels of paclitaxel over time frominhalation study.

FIG. 54 is a graph of lung tissue levels of paclitaxel over time frominhalation study.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. “And” as usedherein is interchangeably used with “or” unless expressly statedotherwise.

As used herein, “about” means+/−five percent (5%) of the recited unit ofmeasure.

All embodiments of any aspect of the invention can be used incombination, unless the context clearly dictates otherwise.

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words ‘comprise’, ‘comprising’, and thelike are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense; that is to say, in the sense of“including, but not limited to”. Words using the singular or pluralnumber also include the plural and singular number, respectively.Additionally, the words “herein,” “above,” and “below” and words ofsimilar import, when used in this application, shall refer to thisapplication as a whole and not to any particular portions of theapplication. The compositions and methods for their use can “comprise,”“consist essentially of,” or “consist of” any of the ingredients orsteps disclosed throughout the specification.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While the specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize.

In a first aspect, the invention provides methods for treating a lungdisorder, comprising pulmonary administration to a subject with a lungdisorder of an amount effective of a composition comprising taxaneparticles to treat the lung tumor, wherein the taxane particles compriseat least 95% of the taxane and have a mean particle size (number) ofbetween 0.1 μm and 5 μm.

The inventors have surprisingly discovered that pulmonary administrationof the taxane particles according to the methods of the invention resultin much longer residency times of the taxane in the lungs than waspreviously possible using any other taxane formulation. As shown in theexamples that follow, the taxane remains detectable in lung tissue ofthe subject for at least 96 hours after the administering. In variousfurther embodiments, the taxane remains detectable in lung tissue of thesubject for at least 108, 120, 132, 144, 156, 168, 180, 192, 204, 216,228, 240, 252, 264, 276, 288, 300, 312, 324, or 336 hours after theadministering. Thus, the methods can be used to treat any lung disorderfor which taxane particles may be an effective treatment, including butnot limited to lung tumors, mesothelioma, restrictive lung diseases suchas pulmonary fibrosis, and obstructive lung diseases such as chronicobstructive lung disease (COPD).

Another aspect of the invention is that the methods also allow forexposure of the taxane particles to a lung tumor after administration ofthe composition for a sustained amount of time sufficient to stimulatethe endogenous immune system of the subject resulting in the productionof tumoricidal cells and infiltration of the tumoricidal cells into thetumor at a level sufficient to treat the tumor. In some embodiments, thestimulation of the endogenous immune systems produces a cellular(cell-mediated) immune response. In other embodiments, the stimulationof the endogenous immune system produces a humoral immune response. Insome embodiments, the stimulation of the endogenous immune systemproduces a tumor vaccine. In some embodiments, metastases are reduced oreliminated. The tumoricidal cells may comprise T-cells, B cells, ornatural killer (NK) cells, or combinations thereof. In some embodiments,the sustained amount of exposure time is at least 108, 120, 132, 144,156, 168, 180, 192, 204, 216, 228, 240, 252, 264, 276, 288, 300, 312,324, or 336 hours. In various further embodiments, the sustained amountof exposure time is at least 3, 4, 5, 6, 7, or 8 weeks. The compositioncan be administered by pulmonary administration in a singleadministration (cycle) of a single dose, or in two or more separateadministrations (2 or more cycles) of single doses. In some embodiments,the two or more separate administrations are administered at or at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 14 days apart. In someembodiments, the two or more separate administrations are administered 2to 12, 2-11, 2-10, 2-9, 2-8 2-7, 2-6, 2-5, 2-4, 2-3, 3-12, 3-11, 3-10,3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5,5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7,7-12, 7-11, 7-10, 7-9, 7-8, 8-12, 8-11, 8-10, 8-9, 9-12, 9-11, 9-10,10-12, 10-11, 11-12, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks apart.In some embodiments, the composition is administered in 2-5, 2-4, 2-3,3-5, 3-4, 2, 3, 4, 5, or more separate administrations. In someembodiments, the two or more separate administrations are administered 2to 12 weeks apart. In some embodiments, the composition is administeredin two to five separate administrations. In some embodiments, the two ormore separate administrations are administered once a week for at leasttwo weeks. In other embodiments, the two or more separateadministrations are administered twice a week for at least one week,wherein the two or more separate administrations are separated by atleast one day. In some embodiments the treatment method results inelimination (eradication) of the tumor. In some embodiments, thecomposition is administered in 1, 2, 3, 4, 5, 6 or more separateadministrations. In other embodiments, the composition is administeredin 7 or more separate administrations.

As used herein, “taxane particles” are particles consisting essentiallyof the taxane (i.e.: at least 95%, 96%, 97%, 98%, 99%, or 100% taxane)that have a mean particle size (number) of between 0.1 μm and 5 μm.Taxane particles for use in the invention are uncoated, and are notembedded, contained, enclosed or encapsulated within a solid excipient.Taxane particles of the invention may, however, contain impurities andbyproducts typically found during preparation of the taxane. Even so,the taxane particles comprise at least 95%, at least 96%, at least 97%,at least 98%, at least 99% or 100% taxane, meaning the taxane particlesconsist of or consist essentially of substantially pure taxane.

Taxanes are a class of diterpenoids containing a taxadiene core that arevery poorly soluble in water. The taxane particles of the invention maybe any suitable taxane, including but not limited to paclitaxel,docetaxel, cabazitaxel, taxadiene, baccatin III, taxchinin A,brevifoliol, and taxuspine D, combinations thereof, or pharmaceuticallyacceptable salts thereof. In one embodiment, the taxane is selected fromthe group consisting of paclitaxel, docetaxel, and cabazitaxel, or apharmaceutically acceptable salt thereof.

Paclitaxel and docetaxel active pharmaceutical ingredients (APIs) arecommercially available from Phyton Biotech LLC, Vancouver, Canada. Thedocetaxel API contains not less than 95%, or not less than 97.5%docetaxel calculated on the anhydrous, solvent-free basis. Thepaclitaxel API contains not less than 95%, or not less than 97%paclitaxel calculated on the anhydrous, solvent-free basis. In someembodiments, the paclitaxel API and docetaxel API are USP and/or EPgrade. Paclitaxel API can be prepared from a semisynthetic chemicalprocess or from a natural source such as plant cell fermentation orextraction.

The lung tumor is any tumor present within the lungs and may be aprimary or a metastatic lung tumor. Non-limiting examples of a lungtumor include small-cell lung carcinoma (SCLC) and non-small-cell lungcarcinoma (NSCLC). In one embodiment, the lung tumor is SCLC. In anotherembodiment, the lung tumor is a NSCLC. The subject may be any mammalsubject to lung tumors, including but not limited to humans and otherprimates, dogs, cats, horses, cattle, pigs, sheep, goats, etc.

The “amount effective” of the taxane particle can be determined by anattending physician based on all relevant factors. The taxane particlesmay be the sole taxane administered, or may be administered with othertherapeutics as deemed appropriate by an attending physician in light ofall circumstances. In one embodiment, the methods further comprisetreating the subject with the standard of care for the tumor beingtreated, such as intravenous chemotherapy, radiation therapy, surgicalresection, etc.

As used herein, “treat”, “treatment”, or “treating” means accomplishingone or more of the following: (a) reducing tumor or fibrosis size; (b)reducing tumor growth rate; (c) eliminating a tumor or fibrosis; (d)reducing or limiting development and/or spreading of metastases, oreliminating metastases. In some embodiments, the treatment iseliminating a tumor or fibrosis.

In one specific embodiment of the invention, pulmonary administrationcomprises inhalation of a single dose of the taxane particles, such asby nasal, oral inhalation, or both. The taxane particles can beadministered in two or more separate administrations (doses). In thisembodiment, the taxane particles may be formulated as an aerosol (i.e.:liquid droplets of a stable dispersion or suspension of theantineoplastic particles in a gaseous medium). Taxane particlesdelivered by aerosol may be deposited in the airways by gravitationalsedimentation, inertial impaction, and/or diffusion. Any suitable devicefor generating the aerosol may be used, including but not limited topressured meter inhalers (pMDI), nebulizers, dry powder inhalers (DPI),and soft-mist inhalers.

In one specific embodiment, the methods comprise inhalation of taxaneparticles aerosolized via nebulization. Nebulizers generally usecompressed air or ultrasonic power to create inhalable aerosol dropletsof the taxane particles or suspensions thereof. In this embodiment, thenebulizing results in pulmonary delivery to the subject of aerosoldroplets of the taxane particles or suspension thereof.

In another embodiment, the methods comprise inhalation of taxaneparticles aerosolized via a pMDI, wherein the taxane particles orsuspensions thereof are suspended in a suitable propellant system(including but not limited to hydrofluoroalkanes (HFAs) containing atleast one liquefied gas in a pressurized container sealed with ametering valve. Actuation of the valve results in delivery of a metereddose of an aerosol spray of the taxane particles or suspensions thereof.

In other embodiments, the taxane particles have a mean particle size(number) greater than 0.2 μm, or 0.3 μm. In another embodiment, thetaxane particles have a mean particle size (number) of at least 0.4 μm.In further embodiments, the taxane particles have a mean particle size(number) of between 0.4 μm and 2 μm, or between 0.5 μm and 1.5 μm, orbetween 0.2 μm and 1 μm, or between 0.2 μm to less than 1 μm.

In further embodiments, the taxane particles can have a mean particlesize number of between in the range of about 0.4 μm to about 5 μm, about0.4 μm to about 3 μm, about 0.5 μm to about 1.4 μm, about 0.4 μm toabout 0.8 μm, about 0.4 μm to about 0.7 μm, or about 0.5 μm to about 0.7μm. In a further embodiment, the taxane particles have a mean particlesize number of between about 0.4 μm and about 1.2 μm, or between about0.6 μm and about 1.0 μm. In another embodiment, the taxane particleshave a mean particle size number of between 0.6 μm and 0.861 μm, orbetween about 0.5 μm to about 0.7 μm, or between about 0.2 μm to about 1μm, or between about 0.2 μm to less than 1 μm, or between about 0.3 μmto about 1 μm, or between about 0.3 μm to less than 1 μm, or betweenabout 0.4 μm to about 1 μm, or between about 0.4 μm to less than 1 μm.

The particle size of the taxane particles can be determined by aparticle size analyzer instrument and the measurement is expressed asthe mean diameter based on a number distribution (number). A suitableparticle size analyzer instrument is one which employs the analyticaltechnique of light obscuration, also referred to as photozone or singleparticle optical sensing (SPOS). A suitable light obscuration particlesize analyzer instrument is the ACCUSIZER, such as the ACCUSIZER 780SIS, available from Particle Sizing Systems, Port Richey, Fla. Anothersuitable particle size analyzer instrument is one which employs laserdiffraction, such as the Shimadzu SALD-7101.

In embodiments where the taxane particles are aerosolized foradministration, the mass median aerodynamic diameter (MMAD) of theaerosol droplets of the taxane particles or suspensions thereof may beany suitable diameter for use in the invention. In one embodiment, theaerosol droplets have a MMAD of between about 0.5 μm to about 6 μmdiameter. In various further embodiments, the aerosol droplets have aMMAD of between about 0.5 μm to about 5.5 μm diameter, about 0.5 μm toabout 5 μm diameter, about 0.5 μm to about 4.5 μm diameter, about 0.5 μmto about 4 μm diameter, about 0.5 μm to about 3.5 μm diameter, about 0.5μm to about 3 μm diameter, about 0.5 μm to about 2.5 μm diameter, about0.5 μm to about 2 μm diameter, about 1 μm to about 5.5 μm diameter,about 1 μm to about 5 μm diameter, about 1 μm to about 4.5 μm diameter,about 1 μm to about 4 μm diameter, about 1 μm to about 3.5 μm diameter,about 1 μm to about 3 μm diameter, about 1 μm to about 2.5 μm diameter,about 1 μm to about 2 μm diameter, about 1.5 μm to about 5.5 μmdiameter, about 1.5 μm to about 5 μm diameter, about 1.5 μm to about 4.5μm diameter, about 1.5 μm to about 4 μm diameter, about 1.5 μm to about3.5 μm diameter, about 1.5 μm to about 3 μm diameter, about 1.5 μm toabout 2.5 μm diameter, about 1.5 μm to about 2 μm diameter, about 2 μmto about 5.5 μm diameter, about 2 μm to about 5 μm diameter, about 2 μmto about 4.5 μm diameter, about 2 μm to about 4 μm diameter, about 2 μmto about 3.5 μm diameter, about 2 μm to about 3 μm diameter, and about 2μm to about 2.5 μm diameter. A suitable instrument for measuring themass median aerodynamic diameter (MMAD) and geometric standard deviation(GSD) of the aerosol droplets is a seven-stage aerosol sampler such asthe Mercer-Style Cascade Impactor.

In another embodiment, the taxane particles may have a specific surfacearea (SSA) of at least 10 m²/g, or at least 12 m²/g, 14 m²/g, 16 m²/g,18 m²/g, 20 m²/g, 25 m²/g, 30 m²/g, 32 m²/g, 34 m²/g, or 35 m²/g; orwherein the taxane particles have an SSA of between about 10 m²/g andabout 60 m²/g.

In various embodiments, the taxane particles are made by “precipitationwith compressed anti-solvents” (PCA) methods as disclosed in U.S. Pat.No. 5,874,029, U.S. Pat. No. 5,833,891, U.S. Pat. No. 6,113,795, U.S.Pat. No. 7,744,923, U.S. Pat. No. 8,778,181, U.S. Pat. No. 9,233,348; USpublications US 2015/0375153, US 2016/0354336, US 2016/0374953; andinternational patent application publications WO 2016/197091, WO2016/197100, and WO 2016/197101; all of which are herein incorporated byreference.

In PCA particle size reduction methods using supercritical carbondioxide, supercritical carbon dioxide (anti-solvent) and solvent, e.g.acetone or ethanol, are employed to generate uncoated taxane particlesas small as 0.1 to 5 microns within a well-characterized particle-sizedistribution. The carbon dioxide and solvent are removed duringprocessing (up to 0.5% residual solvent may remain), leaving taxaneparticles as a powder. Stability studies show that the paclitaxelparticle powder is stable in a vial dose form when stored at roomtemperature for up to 59 months and under accelerated conditions (40°C./75% relative humidity) for up to six months.

Taxane particles produced by various supercritical carbon dioxideparticle size reduction methods can have unique physical characteristicsas compared to taxane particles produced by conventional particle sizereduction methods using physical impacting or grinding, e.g., wet or drymilling, micronizing, disintegrating, comminuting, microfluidizing, orpulverizing. As disclosed in US publication 2016/0374953, hereinincorporated by reference, such unique characteristics include a bulkdensity (the mass of the totality of particles in the compositiondivided by the total volume they occupy when poured into a graduatedcylinder, without tapping the graduated cylinder, with the total volumeincluding particle volume, inter-particle void volume, and internal porevolume.) between 0.05 g/cm³ and 0.15 g/cm³ and a specific surface area(SSA) of at least 18 m²/g of taxane (e.g., paclitaxel and docetaxel)particles, which are produced by the supercritical carbon dioxideparticle size reduction methods described in US publication 2016/0374953and as described below. This bulk density range is generally lower thanthe bulk density of taxane particles produced by conventional means, andthe SSA is generally higher than the SSA of taxane particles produced byconventional means. These unique characteristics result in significantincreases in dissolution rates in water/methanol media as compared totaxanes produced by conventional means. As used herein, the “specificsurface area” (SSA) is the total surface area of the taxane particle perunit of taxane mass as measured by the Brunauer-Emmett-Teller (“BET”)isotherm by the following method: a known mass between 200 and 300 mg ofthe analyte is added to a 30 mL sample tube. The loaded tube is thenmounted to a Porous Materials Inc. SORPTOMETER®, model BET-202A. Theautomated test is then carried out using the BETWIN® software packageand the surface area of each sample is subsequently calculated. As willbe understood by those of skill in the art, the “taxane particles” caninclude both agglomerated taxane particles and non-agglomerated taxaneparticles; since the SSA is determined on a per gram basis it takes intoaccount both agglomerated and non-agglomerated taxane particles in thecomposition. The BET specific surface area test procedure is acompendial method included in both the United States Pharmaceopeia andthe European Pharmaceopeia. The bulk density measurement can beconducted by pouring the taxane particles into a graduated cylinderwithout tapping at room temperature, measuring the mass and volume, andcalculating the bulk density.

As disclosed in US publication 2016/0374953, studies showed a SSA of15.0 m²/g and a bulk density of 0.31 g/cm³ for paclitaxel particlesproduced by milling paclitaxel in a Deco-PBM-V-0.41 ball mill suing a 5mm ball size, at 600 RPM for 60 minutes at room temperature. Alsodisclosed in US publication 2016/0374953, one lot of paclitaxelparticles had a SSA of 37.7 m²/g and a bulk density of 0.085 g/cm³ whenproduced by a supercritical carbon dioxide method using the followingmethod: a solution of 65 mg/mL of paclitaxel was prepared in acetone. ABETE MicroWhirl® fog nozzle (BETE Fog Nozzle, Inc.) and a sonic probe(Qsonica, model number Q700) were positioned in the crystallizationchamber approximately 8 mm apart. A stainless steel mesh filter withapproximately 100 nm holes was attached to the crystallization chamberto collect the precipitated paclitaxel particles. The supercriticalcarbon dioxide was placed in the crystallization chamber of themanufacturing equipment and brought to approximately 1200 psi at about38° C. and a flow rate of 24 kg/hour. The sonic probe was adjusted to60% of total output power at a frequency of 20 kHz. The acetone solutioncontaining the paclitaxel was pumped through the nozzle at a flow rateof 4.5 mL/minute for approximately 36 hours. Additional lots ofpaclitaxel particles produced by the supercritical carbon dioxide methoddescribed above had SSA values of: 22.27 m²/g, 23.90 m²/g, 26.19 m²/g,30.02 m²/g, 31.16 m²/g, 31.70 m²/g, 32.59 m²/g, 33.82 m²/g, 35.90 m²/g,38.22 m²/g, and 38.52 m²/g.

As disclosed in US publication 2016/0374953, studies showed a SSA of15.2 m²/g and a bulk density of 0.44 g/cm³ for docetaxel particlesproduced by milling docetaxel in a Deco-PBM-V-0.41 ball mill suing a 5mm ball size, at 600 RPM for 60 minutes at room temperature. Alsodisclosed in US publication 2016/0374953, docetaxel particles had a SSAof 44.2 m²/g and a bulk density of 0.079 g/cm³ when produced by asupercritical carbon dioxide method using the following method: Asolution of 79.32 mg/mL of docetaxel was prepared in ethanol. The nozzleand a sonic probe were positioned in the pressurizable chamberapproximately 9 mm apart. A stainless steel mesh filter withapproximately 100 nm holes was attached to the pressurizable chamber tocollect the precipitated docetaxel particles. The supercritical carbondioxide was placed in the pressurizable chamber of the manufacturingequipment and brought to approximately 1200 psi at about 38° C. and aflow rate of 68 slpm. The sonic probe was adjusted to 60% of totaloutput power at a frequency of 20 kHz. The ethanol solution containingthe docetaxel was pumped through the nozzle at a flow rate of 2mL/minute for approximately 95 minutes). The precipitated docetaxelagglomerates and particles were then collected from the supercriticalcarbon dioxide as the mixture is pumped through the stainless steel meshfilter. The filter containing the particles of docetaxel was opened andthe resulting product was collected from the filter.

As disclosed in US publication 2016/0374953, dissolution studies showedan increased dissolution rate in methanol/water media of paclitaxel anddocetaxel particles made by the supercritical carbon dioxide methodsdescribed in US publication 2016/0374953 as compared to paclitaxel anddocetaxel particles made by milling paclitaxel and docetaxel using aDeco-PBM-V-0.41 ball mill suing a 5 mm ball size, at 600 RPM for 60minutes at room temperature. The procedures used to determine thedissolution rates are as follows. For paclitaxel, approximately 50 mg ofmaterial were coated on approximately 1.5 grams of 1 mm glass beads bytumbling the material and beads in a vial for approximately 1 hour.Beads were transferred to a stainless steel mesh container and placed inthe dissolution bath containing methanol/water 50/50 (v/v) media at 37°C., pH 7, and a USP Apparatus II (Paddle), operating at 75 rpm. At 10,20, 30, 60, and 90 minutes, a 5 mL aliquot was removed, filtered througha 0.22 μm filter and analyzed on a UV/VIS spectrophotometer at 227 nm.Absorbance values of the samples were compared to those of standardsolutions prepared in dissolution media to determine the amount ofmaterial dissolved. For docetaxel, approximately 50 mg of material wasplaced directly in the dissolution bath containing methanol/water 15/85(v/v) media at 37° C., pH 7, and a USP Apparatus II (Paddle), operatingat 75 rpm. At 5, 15, 30, 60, 120 and 225 minutes, a 5 mL aliquot wasremoved, filtered through a 0.22 μm filter, and analyzed on a UV/VISspectrophotometer at 232 nm. Absorbance values of the samples werecompared to those of standard solutions prepared in dissolution media todetermine the amount of material dissolved. For paclitaxel, thedissolution rate was 47% dissolved in 30 minutes for the particles madeby the supercritical carbon dioxide method versus 32% dissolved in 30minutes for the particles made by milling. For docetaxel, thedissolution rate was 27% dissolved in 30 minutes for the particles madeby the supercritical carbon dioxide method versus 9% dissolved in 30minutes for the particles made by milling.

In some embodiments, the taxane particles have a SSA of at least 10, atleast 12, at least 14, at least 16, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 26, at least 27, at least 28, at least 29, at least 30, at least31, at least 32, at least 33, at least 34, or at least 35 m²/g. In oneembodiment, the antineoplastic particles have an SSA of between about 10m²/g and about 50 m²/g. In some embodiments, the antineoplasticparticles have a bulk density (not tapped) between about 0.050 g/cm³ andabout 0.20 g/cm³.

In further embodiments, the antineoplastic particles have a SSA of:

(a) between 16 m²/g and 31 m²/g or between 32 m²/g and 40 m²/g;

(b) between 16 m²/g and 30 m²/g or between 32 m²/g and 40 m²/g;

(c) between 16 m²/g and 29 m²/g or between 32 m²/g and 40 m²/g;

(d) between 17 m²/g and 31 m²/g or between 32 m²/g and 40 m²/g;

(e) between 17 m²/g and 30 m²/g or between 32 m²/g and 40 m²/g;

(f) between 17 m²/g and 29 m²/g, or between 32 m²/g and 40 m²/g;

(g) between 16 m²/g and 31 m²/g or between 33 m²/g and 40 m²/g;

(h) between 16 m²/g and 30 m²/g or between 33 m²/g and 40 m²/g;

(i) between 16 m²/g and 29 m²/g or between 33 m²/g and 40 m²/g;

(j) between 17 m²/g and 31 m²/g or between 33 m²/g and 40 m²/g;

(k) between 17 m²/g and 30 m²/g or between 33 m²/g and 40 m²/g;

(l) between 17 m²/g and 29 m²/g, or between 33 m²/g and 40 m²/g;

(m) between 16 m²/g and 31 m²/g, or 32 m²/g;

(h) between 17 m²/g and 31 m²/g, or 32 m²/g;

(i) between 16 m²/g and 30 m²/g, or 32 m²/g;

(j) between 17 m²/g and 30 m²/g, or 32 m²/g;

(k) between 16 m²/g and 29 m²/g, or 32 m²/g;

(l) between 17 m²/g and 29 m²/g, or 32 m²/g;

(m) between 16 m²/g and 31 m²/g, or 33 m²/g;

(n) between 17 m²/g and 31 m²/g, or 33 m²/g;

(o) between 16 m²/g and 30 m²/g, or 33 m²/g;

(p) between 17 m²/g and 30 m²/g, or 33 m²/g;

(q) between 16 m²/g and 29 m²/g, or 33 m²/g; or

(r) between 17 m²/g and 29 m²/g, or 33 m²/g.

In some embodiments, the taxane particles are paclitaxel particles andhave an SSA of at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, at least 25, at least 26, at least27, at least 28, at least 29, at least 30, at least 31, at least 32, atleast 33, at least 34, or at least 35 m²/g. In other embodiments, thepaclitaxel particles have an SSA of 18 m²/g to 50 m²/g, or 20 m²/g to 50m²/g, or 22 m²/g to 50 m²/g, or 25 m²/g to 50 m²/g, or 26 m²/g to 50m²/g, or 30 m²/g to 50 m²/g, or 35 m²/g to 50 m²/g, or 18 m²/g to 45m²/g, or 20 m²/g to 45 m²/g, or 22 m²/g to 45 m²/g, or 25 m²/g to 45m²/g, or 26 m²/g to 45 m²/g or 30 m²/g to 45 m²/g, or 35 m²/g to 45m²/g, or 18 m²/g to 40 m²/g, or 20 m²/g to 40 m²/g, or 22 m²/g to 40m²/g, or 25 m²/g to 40 m²/g, or 26 m²/g to 40 m²/g, or 30 m²/g to 40m²/g, or 35 m²/g to 40 m²/g.

In some embodiments, the paclitaxel particles have a bulk density(not-tapped) of 0.05 g/cm³ to 0.15 g/cm³, or 0.05 g/cm³ to 0.20 g/cm³.

In some embodiments, the paclitaxel particles have a dissolution rate ofat least 40% w/w dissolved in 30 minutes or less in a solution of 50%methanol/50% water (v/v) in a USP II paddle apparatus operating at 75RPM, at 37° C., and at a pH of 7.

In another embodiment, the paclitaxel particles have one or more of thefollowing characteristics:

(a) a mean bulk density (not tapped) between about 0.050 g/cm³ and about0.12 g/cm³, or between about 0.060 g/cm³ and about 0.11 g/cm³;

(b) a SSA of at least 12 m²/g, 15 m²/g, 18 m²/g, 20 m²/g, 25 m²/g, 30m²/g, 32 m²/g, 34 m²/g, or 35 m²/g;

(c) a SSA of between about 22 m²/g and about 40 m²/g, 25 m²/g and about40 m²/g, 30 m²/g and about 40 m²/g, or between about 35 m²/g and about40 m²/g; and/or

(d) wherein at least 40% (w/w) of the paclitaxel is dissolved in 30minutes or less in a solution of 50% methanol/50% water (v/v) at 37° C.and pH 7.0 in a USP II paddle apparatus operating at 75 RPM.

In one embodiment, the paclitaxel particles have a mean bulk density(not tapped) of between about between about 0.050 g/cm³ and about 0.12g/cm³ and a SSA of at least 30 m²/g. In another embodiment, thepaclitaxel particles have a mean bulk density (not tapped) of betweenabout between about 0.050 g/cm³ and about 0.12 g/cm³ and a SSA of atleast 35 m²/g. In one embodiment the paclitaxel particles have a meanbulk density (not tapped) of between about between about 0.050 g/cm³ andabout 0.12 g/cm³ and a SSA of between about 30 m²/g and about 40 m²/g.In another embodiment, the paclitaxel particles have a mean bulk density(not tapped) of between about 0.060 g/cm³ and about 0.11 g/cm³ and a SSAof between about 30 m²/g and about 40 m²/g. In another embodiment, thepaclitaxel particles have a mean bulk density (not tapped) of betweenabout 0.060 g/cm³ and about 0.11 g/cm³ and a SSA of at least 30 m²/g. Ina further embodiment, the paclitaxel particles have a mean bulk density(not tapped) of between about 0.060 g/cm³ and about 0.11 g/cm³ and a SSAof at least 35 m²/g.

In another embodiment, at least 40% (w/w) of the paclitaxel in thepaclitaxel particles of the composition is dissolved in 30 minutes orless in a solution of 50% methanol/50% water (v/v) in a USP II paddleapparatus operating at 75 RPM. pH 7 was used, and the solubility of thetaxanes are not effected by pH. In another embodiment, the dissolutionstudies are carried out at 37° C.

In some embodiments, the taxane particles are docetaxel particles andhave an SSA of at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, at least 25, at least 26, at least27, at least 28, at least 29, at least 30, at least 31, at least 32, atleast 33, at least 34, at least 35, at least 36, at least 37, at least38, at least 39, at least 40, at least 41, or at least 42 m²/g. In otherembodiments, the docetaxel particles have an SSA of 18 m²/g to 60 m²/g,or 22 m²/g to 60 m²/g, or 25 m²/g to 60 m²/g, or 30 m²/g to 60 m²/g, or40 m²/g to 60 m²/g, or 18 m²/g to 50 m²/g, or 22 m²/g to 50 m²/g, or 25m²/g to 50 m²/g, or 26 m²/g to 50 m²/g, or 30 m²/g to 50 m²/g, or 35m²/g to 50 m²/g, or 40 m²/g to 50 m²/g.

In some embodiments, the docetaxel particles have a bulk density(not-tapped) of 0.05 g/cm³ to 0.15 g/cm³.

In some embodiments, the docetaxel particles have a dissolution rate ofat least 20% w/w dissolved in 30 minutes or less in a solution of 15%methanol/85% water (v/v) in a USP II paddle apparatus operating at 75RPM, at 37° C., and at a pH of 7.

In another embodiment, the docetaxel particles have one or more of thefollowing characteristics:

(a) a mean bulk density (not tapped) between about 0.050 g/cm³ and about0.12 g/cm³, or between about 0.06 g/cm³ and about 0.1 g/cm³;

(b) a SSA of at least 12 m²/g, 15 m²/g, 18 m²/g, 20 m²/g, 25 m²/g, 30m²/g, 35 m²/g, 40 m²/g, or 42 m²/g;

(c) a SSA of between about 20 m²/g and about 50 m²/g, or between about35 m²/g and about 46 m²/g; and/or

(d) wherein at least 20% (w/w) of the docetaxel is dissolved in 30minutes or less in a solution of 15% methanol/85% water (v/v) at 37° C.and pH 7.0 in a USP II paddle apparatus operating at 75 RPM.

In one embodiment, the docetaxel particles have a mean bulk density (nottapped) between about 0.050 g/cm³ and about 0.12 g/cm³ and a SSA of atleast 30 m²/g. In another embodiment, the docetaxel particles have amean bulk density (not tapped) between about 0.050 g/cm³ and about 0.12g/cm³ and a SSA of at least 35 m²/g. In a further embodiment, thedocetaxel particles have a mean bulk density (not tapped) between about0.050 g/cm³ and about 0.12 g/cm³ and a SSA of at least 40 m²/g. In oneembodiment, the docetaxel particles have a mean bulk density (nottapped) between about 0.050 g/cm³ and about 0.12 g/cm³ and a SSA ofbetween about 20 m²/g and about 50 m²/g. In another embodiment, meanbulk density (not tapped) of the docetaxel particles is between about0.06 g/cm³ and about 0.1 g/cm³ and the SSA is between about 30 m²/g andabout 50 m²/g. In another embodiment, mean bulk density (not tapped) ofthe docetaxel particles is between about 0.06 g/cm³ and about 0.1 g/cm³and the SSA is between about 35 m²/g and about 50 m²/g. In anotherembodiment, mean bulk density (not tapped) of the docetaxel particles isbetween about 0.06 g/cm³ and about 0.1 g/cm³ and the SSA is betweenabout 35 m²/g and about 45 m²/g.

In another embodiment, at least 20% (w/w) of the docetaxel is dissolvedin 30 minutes or less in a solution of 15% methanol/85% water (v/v) in aUSP II paddle apparatus operating at 75 RPM. A neutral pH was used wherethe solubility of the taxanes are not effected by pH. In anotherembodiment, the dissolution studies are carried out at 37° C.

In any of these various embodiments, the taxane particles may include atleast 4.16×10⁻¹³ gram taxane, or a pharmaceutically acceptable saltthereof per taxane particle. In some embodiments, the taxane particlesare non-agglomerated individual particles and are not clusters ofmultiple taxane particles.

In various embodiments of the present invention, the taxane particlesare uncoated (neat) individual particles; the taxane particles are notbound to or conjugated to any substance; no substances are absorbed oradsorbed onto the surface of the taxane particles; the taxane particlesare not encapsulated in any substance; the taxane particles are notcoated with any substance; the taxane particles are not microemulsions,nanoemulsions, microspheres, or liposomes of a taxane; and/or the taxaneparticles are not bound to, attached to, encapsulated in, or coated witha monomer, a polymer (or biocompatible polymer), a protein, asurfactant, or albumin. In some embodiments, a monomer, a polymer (orbiocompatible polymer), a copolymer, a protein, a surfactant, or albuminis not absorbed or adsorbed onto the surface of the taxane particles. Insome embodiments, the compositions are free of/do not include or containa polymer/copolymer or biocompatible polymer/copolymer. In someembodiments, the compositions are free of/do not include or contain aprotein. In some aspects of the invention, the compositions are freeof/do not include or contain albumin. In some aspects of the invention,the compositions are free of/do not include or contain hyaluronic acid.In some aspects of the invention, the compositions are free of/do notinclude or contain a conjugate of hyaluronic acid and a taxane. In someaspects of the invention, the compositions are free of/do not include orcontain a conjugate of hyaluronic acid and paclitaxel. In some aspectsof the invention, the compositions are free of/do not include or containpoloxamers, polyanions, polycations, modified polyanions, modifiedpolycations, chitosan, chitosan derivatives, metal ions, nanovectors,poly-gamma-glutamic acid (PGA), polyacrylic acid (PAA), alginic acid(ALG), Vitamin E-TPGS, dimethyl isosorbide (DMI), methoxy PEG 350,citric acid, anti-VEGF antibody, ethylcellulose, polystyrene,polyanhydrides, polyhydroxy acids, polyphosphazenes, polyorthoesters,polyesters, polyamides, polysaccharides, polyproteins,styrene-isobutylene-styrene (SIBS), a polyanhydride copolymer,polycaprolactone, polyethylene glycol (PEG), Poly(bis(P-carboxyphenoxy)propane-sebacic acid, poly(d,l-lactic acid) (PLA),poly(d.l-lactic acid-co-glycolic acid) (PLAGA), and/or poly(D, Llactic-co-glycolic acid (PLGA). In some embodiments, the taxaneparticles are in crystalline form. In other embodiments, the taxaneparticles are in amorphous form, or a combination of both crystallineand amorphous form.

In one embodiment, the taxane particles for administration comprises adosage form of taxane in suspension (i.e.: with a pharmaceuticallyacceptable carrier, and or in an aerosol formulation) of between about0.1 mg/ml and about 100 mg/ml taxane. In various further embodiments,the dosage form may be between about 0.5 mg/ml and about 100 mg/ml,about 1 mg/ml and about 100 mg/ml, about 2 mg/ml and about 100 mg/ml,about 5 mg/ml and about 100 mg/ml, about 10 mg/ml and about 100 mg/ml,about 25 mg/ml and about 100 mg/ml, about 0.1 mg/ml and about 75 mg/ml,about 0.5 mg/ml and about 75 mg/ml, about 1 mg/ml and about 75 mg/ml,about 2 mg/ml and about 75 mg/ml, about 5 mg/ml and about 75 mg/ml,about 10 mg/ml and about 75 mg/ml, about 25 mg/ml and about 75 mg/m,about 0.1 mg/ml and about 50 mg/ml, about 0.5 mg/ml and about 50 mg/ml,about 1 mg/ml and about 50 mg/ml, about 2 mg/ml and about 50 mg/ml,about 5 mg/ml and about 50 mg/ml, about 10 mg/ml and about 50 mg/ml,about 25 mg/ml and about 50 mg/m, about 0.1 mg/ml and about 25 mg/ml,about 0.5 mg/ml and about 25 mg/ml, about 1 mg/ml and about 40 mg/ml,about 1 mg/ml and about 25 mg/ml, about 2 mg/ml and about 25 mg/ml,about 5 mg/ml and about 25 mg/ml, about 10 mg/ml and about 25 mg/ml,about 0.1 mg/ml and about 15 mg/ml, about 0.5 mg/ml and about 15 mg/ml,about 1 mg/ml and about 15 mg/ml, about 2 mg/ml and about 15 mg/ml,about 5 mg/ml and about 15 mg/ml, about 10 mg/ml and about 15 mg/ml,about 0.1 mg/ml and about 10 mg/ml, about 0.5 mg/ml and about 10 mg/ml,about 1 mg/ml and about 10 mg/ml, about 2 mg/ml and about 10 mg/ml,about 5 mg/ml and about 10 mg/ml, about 0.1 mg/ml and about 5 mg/ml,about 0.5 mg/ml and about 5 mg/ml, about 1 mg/ml and about 5 mg/ml,about 2 mg/ml and about 5 mg/ml, about 0.1 mg/ml and about 2 mg/ml,about 0.5 mg/ml and about 2 mg/ml, about 1 mg/ml and about 2 mg/ml,about 0.1 mg/ml and about 1 mg/ml, about 0.5 mg/ml and about 1 mg/ml,about 0.1 mg/ml and about 0.5 mg/ml, about 0.1 mg/ml and about 15 mg/ml,about 0.5 mg/ml and about 15 mg/ml, about 1 mg/ml and about 15 mg/ml,about 2 mg/ml and about 15 mg/ml, about 5 mg/ml and about 15 mg/ml,about 3 mg/ml and about 8 mg/ml, or about 4 mg/ml and about 6 mg/mltaxane, or at least about 0.1, 0.5, 1, 10, 20, 25, 50, 75, or 100 mg/mltaxane.

In one embodiment, the taxane particles are present in a liquid carrierprior to aerosolization. Any suitable liquid carrier may be used, suchas an aqueous liquid carrier. Any suitable aqueous liquid carrier can beused, including but not limited to 0.9% saline solution (normal saline)such as 0.9% Sodium Chloride for Injection USP. In another embodiment,the taxane particles are present in a suspension prior toaerosolization. In some embodiments, the suspension includes an aqueouscarrier. The carrier can comprise buffering agent, osmotic salt and/orsurfactant in water, and other agents for adjustment of pH, isotonicityand viscosity. In one embodiment employing an aqueous carrier, theconcentration of surfactant is less than 1% on a w/w or w/v basis; inother embodiments, less than 0.5%, less than 0.25%, or about 0.1%. Inother embodiments, the aqueous carrier can exclude the surfactantsGELUCIRE® (polyethylene glycol glycerides composed of mono-, di- andtriglycerides and mono- and diesters of polyethylene glycol) and/orCREMOPHOR® (polyethoxylated castor oil). In some embodiments, thecomposition or suspension excludes polymers, proteins (such as albumin),polyethoxylated castor oil, and/or polyethylene glycol glyceridescomposed of mono-, di- and triglycerides and mono- and diesters ofpolyethylene glycol.

In some embodiments, the suspension can comprise water and optionallyone or more excipients selected from the group consisting of buffer,tonicity adjusting agent, preservative, demulcent, viscosity modifier,osmotic agent, surfactant, antioxidant, alkalinizing agent, acidifyingagent, antifoaming agent, and colorant. For example, the suspension cancomprise taxane particles, water, buffer and salt. It optionally furthercomprises a surfactant. In some embodiments, the suspension consistsessentially of or consists of water, paclitaxel particles suspended inthe water and buffer. The suspension can further contain an osmoticsalt.

The suspension can comprise one or more surfactants. Suitablesurfactants include by way of example and without limitationpolysorbates, lauryl sulfates, acetylated monoglycerides, diacetylatedmonoglycerides, and poloxamers. Polysorbates are polyoxyethylenesorbitan fatty acid esters which are a series of partial fatty acidesters of sorbitol and its anhydrides copolymerized with approximately20, 5, or 4 moles of ethylene oxide for each mole of sorbitol and itsanhydrides. Non-limiting examples of polysorbates are polysorbate 20,polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61,polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, andpolysorbate 120. Polysorbates containing approximately 20 moles ofethylene oxide are hydrophilic nonionic surfactants. Examples ofpolysorbates containing approximately 20 moles of ethylene oxide includepolysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65,polysorbate 80, polysorbate 85, and polysorbate 120. Polysorbates areavailable commercially from Croda under the tradename TWEEN™. The numberdesignation of the polysorbate corresponds to the number designation ofthe TWEEN, e.g., polysorbate 20 is TWEEN 20, polysorbate 40 is TWEEN 40,polysorbate 60 is TWEEN 60, polysorbate 80 is TWEEN 80, etc. USP/NFgrades of polysorbate include polysorbate 20 NF, polysorbate 40 NF,polysorbate 60 NF, and polysorbate 80 NF. Polysorbates are alsoavailable in PhEur grades (European Pharmacopoeia), BP grades, and JPgrades. The term “polysorbate” is a non-proprietary name. The chemicalname of polysorbate 20 is polyoxyethylene 20 sorbitan monolaurate. Thechemical name of polysorbate 40 is polyoxyethylene 20 sorbitanmonopalmitate. The chemical name of polysorbate 60 is polyoxyethylene 20sorbitan monostearate. The chemical name of polysorbate 80 ispolyoxyethylene 20 sorbitan monooleate. In some embodiments, thesuspension can comprise mixtures of polysorbates. In some embodiments,the suspension comprises polysorbate 20, polysorbate 40, polysorbate 60,polysorbate 65, polysorbate 80, polysorbate 85, and/or polysorbate 120.In other embodiments, the suspension comprises polysorbate 20,polysorbate 40, polysorbate 60, and/or polysorbate 80. In oneembodiment, the suspension comprises polysorbate 80.

The suspension can comprise one or more tonicity adjusting agents.Suitable tonicity adjusting agents include by way of example and withoutlimitation, one or more inorganic salts, electrolytes, sodium chloride,potassium chloride, sodium phosphate, potassium phosphate, sodium,potassium sulfates, sodium and potassium bicarbonates and alkaline earthmetal salts, such as alkaline earth metal inorganic salts, e.g., calciumsalts, and magnesium salts, mannitol, dextrose, glycerin, propyleneglycol, and mixtures thereof.

The suspension can comprise one or more buffering agents. Suitablebuffering agents include by way of example and without limitation,dibasic sodium phosphate, monobasic sodium phosphate, citric acid,sodium citrate hydrochloric acid, sodium hydroxide,tris(hydroxymethyl)aminomethane,bis(2-hydroxyethyl)iminotris-(hydroxymethyl)methane, and sodium hydrogencarbonate and others known to those of ordinary skill in the art.Buffers are commonly used to adjust the pH to a desirable range forintraperitoneal use. Usually a pH of around 5 to 9, 5 to 8, 6 to 7.4,6.5 to 7.5, or 6.9 to 7.4 is desired.

The suspension can comprise one or more demulcents. A demulcent is anagent that forms a soothing film over a mucous membrane, such as themembranes lining the peritoneum and organs therein. A demulcent mayrelieve minor pain and inflammation and is sometimes referred to as amucoprotective agent. Suitable demulcents include cellulose derivativesranging from about 0.2 to about 2.5% such as carboxymethylcellulosesodium, hydroxyethyl cellulose, hydroxypropyl methylcellulose, andmethylcellulose; gelatin at about 0.01%; polyols in about 0.05 to about1%, also including about 0.05 to about 1%, such as glycerin,polyethylene glycol 300, polyethylene glycol 400, and propylene glycol;polyvinyl alcohol from about 0.1 to about 4%; povidone from about 0.1 toabout 2%; and dextran 70 from about 0.1% when used with anotherpolymeric demulcent described herein.

The suspension can comprise one or more alkalinizing agents to adjustthe pH. As used herein, the term “alkalizing agent” is intended to meana compound used to provide an alkaline medium. Such compounds include,by way of example and without limitation, ammonia solution, ammoniumcarbonate, potassium hydroxide, sodium carbonate, sodium bicarbonate,and sodium hydroxide and others known to those of ordinary skill in theart

The suspension can comprise one or more acidifying agents to adjust thepH. As used herein, the term “acidifying agent” is intended to mean acompound used to provide an acidic medium. Such compounds include, byway of example and without limitation, acetic acid, amino acid, citricacid, nitric acid, fumaric acid and other alpha hydroxy acids,hydrochloric acid, ascorbic acid, and nitric acid and others known tothose of ordinary skill in the art.

The suspension can comprise one or more antifoaming agents. As usedherein, the term “antifoaming agent” is intended to mean a compound orcompounds that prevents or reduces the amount of foaming that forms onthe surface of the fill composition. Suitable antifoaming agents includeby way of example and without limitation, dimethicone, SIMETHICONE,octoxynol and others known to those of ordinary skill in the art.

The suspension can comprise one or more viscosity modifiers thatincrease or decrease the viscosity of the suspension. Suitable viscositymodifiers include methylcellulose, hydroxypropyl methycellulose,mannitol and polyvinylpyrrolidone.

In some embodiments, the taxane particle is present in a suspensionfurther comprising a polysorbate. In one specific embodiment, the taxaneparticle is present in a suspension further comprising a polysorbate,wherein the polysorbate is polysorbate 80. In other embodiments, thepolysorbate or polysorbate 80 is present in the suspension at aconcentration of between about 0.01% v/v and about 1.5% v/v. Theinventors have surprisingly discovered that the recited very smallamounts of polysorbate 80 reduce the surface tension at the interface ofthe taxane particles and the aqueous carrier in the suspension (such assaline). In some embodiments, the particles may be coated with thepolysorbate or polysorbate 80, in other embodiments the particles arenot coated with the polysorbate or polysorbate 80. In various otherembodiments, the polysorbate or polysorbate 80 is present in thesuspension at a concentration of between about 0.01% v/v and about 1%v/v, about 0.01% v/v and about 0.5% v/v, about 0.01% v/v and about 0.4%v/v, about 0.01% v/v and about 0.25% v/v, about 0.05% v/v and about 0.5%v/v, about 0.05% v/v and about 0.25% v/v, about 0.1% v/v and about 0.5%v/v, about 0.1% v/v and about 0.25% v/v, about 0.1% v/v, about 0.16 v/v,or about 0.25% v/v. In further embodiments, the taxane, such aspaclitaxel, is present in the suspension at a concentration betweenabout 1 mg/ml and about 40 mg/ml, or about 6 mg/ml and about 20 mg/ml.In various further embodiments, the taxane is present in the suspensionat a concentration between about 6 mg/ml and about 15 mg/ml, betweenabout 6 mg/ml and about 10 mg/ml, about 10 mg/ml and about 20 mg/ml,about 10 mg/ml and about 15 mg/ml, about 6 mg/ml, about 10 mg/ml, orabout 15 mg/ml. In various further embodiments, the aqueous carrier inthe composition may be saline, such as about 0.9% sodium chloride.

Example 1 paclitaxel particles (i.e.: paclitaxel particles as disclosedherein, approximately 98% paclitaxel with a mean particle size (number)of 0.83 microns, a SSA of 27.9 m²/g, and a bulk density (not tapped) of0.0805 g/cm³ used in examples 1, 2, 3, and 4) in Suspension—Safety andEfficacy Development Program—Pilot Pharmacokinetic Study in SpragueDawley Rats

Study Number: FY17-008A

Executive Summary

The objective of this pilot study was to define sampling time points fora complete pharmacokinetic (PK) study with paclitaxel particlesuspension formulation. Due to the potential for the paclitaxel particleformulation to result in increased retention in the lungs, nine timepoints from 0.5 to 168 hours were evaluated to determine the appropriatesampling strategy for a complete pharmacokinetic study.

Sixteen (16) Sprague Dawley rats were exposed to paclitaxel particleformulation (target dose of 0.37 mg/kg) by nose only inhalation on asingle occasion. Two animals (n=2) were euthanatized at their designatedtime point of 0.5, 6, 12, 24, 48, 72, 120 and 168 hours post exposure.Samples of blood (plasma) and lung tissue were collected.

On the day of exposure, the paclitaxel particle formulation (6 mg/mL)was prepared as per instructions provided by the sponsor

Total aerosol exposure time was 63 minutes for all animals. Aerosolconcentration was monitored throughout the 63 minute paclitaxel particleformulation aerosol exposure by measuring the amount of formulationaccumulated on 47-mm GF/A filters positioned at the breathing zone in anose-only exposure chamber. The aerosol particle size (droplet size) wasmeasured using Mercer style cascade impactor from animal breathing zoneon the exposure chamber.

paclitaxel particle suspension formulation was aerosolized using twoHospitak compressed air jet nebulizers (average Paclitaxel aerosolconcentration: target 82.65 μg/L). The overall average aerosolconcentration as measured from the GF/A filters was 0.24 mg/L, and theaverage Paclitaxel aerosol concentration was 73.5 μg/mL. The particlesize distribution was measured to be 2.0 μm MMAD with a GSD of 2.2. Themeasured average Paclitaxel aerosol concentration of 73.5 μg/L was ˜11%lower from target average Paclitaxel aerosol concentration of 82.65 μg/L(within the accuracy/recovery performance criteria of the analyticalassay of ±15%). Oxygen and temperature were monitored throughout thepaclitaxel particles formulation aerosols exposure. The recorded oxygenand temperature ranges were 19.7%-20.9% and 20.4° C.-20.8° C.,respectively.

Paclitaxel deposited dose to the lung was calculated based on thePaclitaxel average aerosol concentration of 73.5 μg/L, average rodentbody weight of 326 g, assumed deposition fraction of 10% and exposureduration of 63 minutes. The average achieved rodent deposited dose wasdetermined to be 0.33 mg/kg. The average achieved deposited dose was˜11% lower when compared to target deposited dose of 0.37 mg/kg, but waswithin the expected variability (±15% from target) for nebulizedexposures.

All animals survived to their designated necropsy timepoint. Atnecropsy, several animals had minimal, red discolorations on the lungs.No other abnormal gross observations were noted at necropsy. From bodyand lung weights obtained at necropsy, average terminal bodyweight amonganimals at all timepoints (standard deviation) was 346.26 g (24.01); andaverage lung weight (standard deviation) was 1.60 g (0.13).

Systemic blood (in the form of plasma from K₂EDTA) was assayed via theliquid chromatography-mass spectrometry (LCMS) assay and lung tissue wasassayed as briefly described in section 4.6 (Bioanalytical Analysis) toquantify the amount of paclitaxel as a function of time. The lung tissueanalysis showed lung exposure with detectable amounts of Paclitaxel outto 168 hours. The systemic blood showed no detectable Paclitaxel (under1 ng/mL) after 24 hours. Based on these data the following samplingtimepoints are suggested for the PK study: 0.5 (±10 minutes), 6 (±10minutes), 12 (±10 minutes), 24 (±30 minutes), 48 (±30 minutes), 72 (±30minutes), 120 (±30 minutes) 168 (±30 minutes), 240 (±30 minutes) and 336(±30 minutes) post inhalation exposure.

Objectives

The objective of this pilot study was to define sampling timepoints fora complete pharmacokinetic (PK) study with paclitaxel particleformulation. The preliminary data with paclitaxel particle formulationdosed by intraperitoneal (IP) injection indicate a significant retentiontime in the intraperitoneal cavity. Due to the potential for thepaclitaxel particles formulation to result in increased retention in thelungs, time points out to 168 hours were evaluated to determine theappropriate sampling strategy for a complete pharmacokinetic study.

Materials and Methods Test System

-   Species/Strain: Sprague Dawley Rats-   Age of Animals at Study Start: 8-10 weeks of age-   Body Weight Range at Study Start: 308-353 g-   Number on Study/Sex: 18 Males (16 study animals and 2 spares)-   Source: Charles River Laboratories (Kingston, N.Y.)-   Identification: Permanent maker tail marking

Test and Control Article Formulation and Administration

A paclitaxel particle suspension formulation (6 mg/mL) was prepared asper instructions provided by the sponsor. Briefly, 5.0 mL of 1%Polysorbate 80 was added to the vial containing paclitaxel particles(306 mg). The paclitaxel particle suspension vial was shaken vigorouslyand inverted to ensure wetting of all particles present in the vial.Immediately after shaking, 46 mL of 0.9% Sodium Chloride was added tothe vial and the vial was shaken for at least 1 minute to make suresufficient mixing and proper dispersion of suspension. Resultantformulation was left undisturbed for at least 5 minutes to reduce anyair/foam in the vial before placing it in the nebulizer foraerosolization work. The final formulation was kept at room temperatureand used within 3 hours after reconstitution.

Experimental Design

Sixteen (16) Sprague Dawley rats were exposed to the paclitaxel particlesuspension formulation (target dose of 0.37 mg/kg) by nose onlyinhalation on a single occasion. Two animals (n=2) were euthanatized at0.5 (±10 minutes), 6 (±10 minutes), 12 (±10 minutes), 24 (±30 minutes),48 (±30 minutes), 72 (±30 minutes), 120 (±30 minutes) and 168 (±30minutes) hours post exposure for blood (plasma) and lung tissuecollections. No specific PK modeling was done; rather, data will definethe duration for detectable amounts of paclitaxel post exposure for thePK Study.

Husbandry, Quarantine and Assignment to Study

Male Sprague Dawley rats (6-8 weeks old) were obtained from CharlesRiver Laboratories (Kingston, N.Y.) and quarantined for 14 days. At theend of quarantine, animals were weighed and then randomized by weightfor assignment to study. Animals were identified by tail marking andcage card. Water, lighting, humidity, and temperature control weremaintained and monitored using standard techniques. Rats were fed astandard rodent diet ad libitum during non-exposure hours.

Body Weights and Daily Observations

Body weights were collected at randomization, daily throughout the studyand at euthanasia. Each animal on study was observed twice daily byComparative Medicine Animal Resources (CMAR) personnel for any clinicalsigns of abnormality, moribundity or death.

Nose-Only Aerosol Exposures Conditioning

Animals were conditioned to nose-only exposure tubes for up to 70minutes using standard techniques. Three conditioning sessions occurredover three days prior to exposure, with the first session lasting 30minutes, the second 60 minutes and the third 70 minutes. They weremonitored closely throughout the conditioning periods and duringexposures to assure that they did not experience more than momentarydistress.

Exposure System

The inhalation exposure system consisted of two compressed air jetnebulizer (Hospitak) and a rodent nose-only inhalation exposure chamber.Exposure oxygen levels (%) were monitored throughout the exposure. Apaclitaxel particle suspension formulation aerosol was generated with aset of two compressed air jet nebulizers (used for up to 40 (±1)minutes, then replaced with a second set of two compressed air jetnebulizers for remaining exposure duration) with an inlet pressure of 20psi. The aerosol was directed through a 24-inch stainless steel aerosoldelivery line (with a 1.53 cm diameter) into a nose-only exposurechamber.

Concentration Monitoring

Aerosol concentration monitoring was conducted by collecting aerosolsonto pre-weighed GF/A 47-mm filters. The filters were sampled fromrodent breathing zones of the nose-only exposure chamber throughout therodent exposure. The aerosol sampling flow rate through GF/A filterswere maintained at 1.0±0.5 L/minute. A total of six GF/A filters werecollected, one every 10 minutes throughout the exposure duration with anexception of the last filter which was collected after 13 minutes. Aftersample collection, filters were weighed to determine the total aerosolconcentration in the exposure system. The filters were extracted andanalyzed by high performance liquid chromatography (HPLC) to quantifythe amount of Paclitaxel collected on each filter. The total aerosolconcentration and Paclitaxel aerosol concentrations were calculated foreach filter by dividing the total amount of aerosols and Paclitaxelaerosols collected with total air flow through the filter. The averagePaclitaxel aerosol concentration was used to calculate the achievedaverage deposited dose of Paclitaxel to the rodent lungs using equation1 as shown below.

Particle Size Determination

Particle size distribution of aerosols was measured from rodentbreathing zone of the nose-only exposure chamber by a Mercer-style,seven-stage cascade impactor (Intox Products, Inc., Albuquerque, N.Mex.). The particle size distribution was determined in terms of massmedian aerodynamic diameter (MMAD) and geometric standard deviation(GSD). Cascade impactor sample was collected at a flow rate of 2.0±0.1L/min.

Determination of Dose

Deposited dose was calculated using Equation 1. In this calculation theaverage aerosol concentration measured from the exposures along withaverage group body weights for rats were used. In this manner theestimated amount of Paclitaxel that was deposited in the rat lungs wascalculated using the measured Paclitaxel aerosol concentration.

$\begin{matrix}{{{DD}\left( {{\mu g}\text{/}{kg}} \right)} = \frac{A\; {C\left( {{\mu g}\text{/}L} \right)} \times {{RMV}\left( {L\text{/}{\min.}} \right)} \times {DF} \times {T\left( {\min.} \right)}}{{BW}({kg})}} & {{Eqn}.\mspace{14mu} 1}\end{matrix}$

Where:

Deposited Dose=(DD) μg/kg

²Respiratory minute volume (RMV)=0.608×BW^(0.852)

Aerosol exposure concentration (AC)=Paclitaxel aerosol concentration(μg/L)

Deposition Fraction (DF)=assumed deposition fraction of 10%

BW=average body weight (at randomization; Day −1) of animals on study(kg)

Euthanasia and Necropsy

Animals were euthanized at their respective time points by an IPinjection of euthanasia solution. During necropsy, blood (for plasma)was collected by cardiac puncture into K₂EDTA tube, lungs were weighed,lung tissue samples were collected and snap frozen in liquid nitrogenfor bioanalytical analyses. Additionally, a full gross examination wasperformed by qualified necropsy personnel. External surfaces of thebody, orifices, and the contents of the cranial, thoracic, and abdominalcavities were examined. Lesions were described and recorded using a setof glossary terms for morphology, quantity, shape, color, consistency,and severity.

Bioanalytical Analyses

Systemic blood (in the form of plasma from K₂EDTA) and lung tissue wereassayed via the liquid chromatography-mass spectrometry (LCMS) assay toquantify the amount of paclitaxel as a function of time. In brief theassay utilizes an ultra-performance liquid chromatography tandem massspectrometry (UPLC-MS/MS) assay to quantify paclitaxel. Plasma samplesare extracted via a protein precipitation method and separation isachieved via reversed phase chromatography. Lung samples werehomogenized with water at a ratio of 4:1 (water:lung tissue). Thehomogenate then underwent a similar protein precipitation method priorto analysis on the LCMS. Quantification was conducted with a matrixbased calibration curve.

No pharmacokinetic modeling was conducted on these data; however, theconcentration at which paclitaxel drops below the sensitivity limits ofthe assay (1 ng/mL) was used to define the sampling timepoints for themain PK study.

Results Clinical Observations and Survival

All animals survived to their designated necropsy timepoint and gainedweight. No abnormal clinical observations were noted through theduration of the study.

Paclitaxel Particle Exposures Aerosol Concentration

Table 1 shows total aerosol and Paclitaxel aerosol concentrationsmeasured by sampling each GF/A filter during exposures. The inhalationexposure average Paclitaxel aerosol concentration of 73.5 μg/L was ˜11%lower from target average Paclitaxel aerosol concentration of 82.65μg/L. The average exposure aerosol concentration was within ±15% oftarget aerosol concentration which was expected for nebulized inhalationexposures.

TABLE 1 Aerosol concentrations during FY17-008A inhalation exposure.Total Aerosol Conc. Paclitaxel Aerosol Filter ID (mg/L) Conc. (μg/L)FS-1 0.230 68.97 FS-2 0.236 71.82 FS-3 0.240 77.58 FS-4 0.268 87.11 FS-50.205 62.11 FS-6 0.237 73.12 Average 0.24 73.5 SD 0.02 8.4 % RSD 8.5511.5

Oxygen and Temperature

The recorded oxygen and temperature ranges were 19.7%-20.9% and 20.4°C.-20.8° C., respectively.

Particle Size

The particle size distribution was determined in terms of MMAD (GSD) for6.0 mg/mL paclitaxel particle formulation aerosols using cascadeimpactor was 2.0 (2.2) μm.

Deposited Dose

Based on Paclitaxel average aerosol concentration of 73.5 μg/L, averagerodent Day −1 (randomization) body weight of 326 g, assumed depositionfraction of 10% and exposure duration of 63 minutes; the averageachieved rodent deposited dose was determined to be 0.33 mg/kg. Theaverage achieved deposited dose was ˜11% lower when compared to targetdeposited dose of 0.37 mg/kg due to expected variability (±15% fromtarget) in exposure average aerosol concentration.

Necropsy

All animals survived to their designated necropsy timepoint. At necropsyseveral animals had minimal, red discolorations on the lungs. No otherabnormal gross observations were noted at necropsy. Individual andaverage lung weights, body weights and ratios were determined. Averageterminal bodyweight (standard deviation) was 346.26 g (24.01). Averagelung weight (standard deviation) was 1.60 g (0.13). Organ lung weightsand lung weight to body weight ratios are common parameters used toassess potential toxicological responses to inhaled materials. Overall,the data are in line with historical data and indicate that there wasnot a response with either of these endpoints.

Bioanalytical

Results are summarized below in Table 2 and FIG. 1. Average paclitaxelconcentration in plasma was 16.705 ng/mL at 0.5 hours post exposure,then decreased gradually through the 24 hour timepoint and was below thelower limit of quantification (1 ng/mL) for all subsequent timepoints.Average paclitaxel concentration in lung tissue was 21940 ng/g at 0.5hours post exposure and decreased gradually to 419.6 ng/g by the 168hour timepoint. This indicates significant paclitaxel particle retentionin the lung with minimal systemic exposure.

TABLE 2 Lung tissue and plasma results Lung Tissue Plasma Lung TissuePlasma Average Average Conc. Animal Timepoint ConcentrationConcentration Conc. (ng/mL) Per (ng/g) Per Number (hr) (ng/mL) (ng/g)timepoint timepoint 1001 0.5 8.81 16680 16.705  21940 1002 24.6  272001003 6 4.46 7800 4.695 7160 1004 4.93 6520 1005 12 3.72 8240 3.720 63201006 <LLOQ 4400 1007 24 <LLOQ 3144 3.140 4452 1008 3.14 5760 1009 48<LLOQ 2300 <LLOQ 2652 1010 <LLOQ 3004 1011 72 <LLOQ 1760 <LLOQ 2028 1012<LLOQ 2296 1013 120 <LLOQ 608 <LLOQ 486.8 1014 <LLOQ 366 1015 168 <LLOQ572 <LLOQ 419.6 1016 <LLOQ 267

Conclusions

Sixteen (16) male Sprague Dawley rats were exposed to paclitaxelparticle formulation aerosols (target dose of 0.37 mg/kg) by nose onlyinhalation on a single occasion. Two animals (n=2) were euthanatized at0.5, 6, 12, 24, 48, 72, 120 and 168 hours post exposure for blood(plasma) and lung tissue collections.

The average Paclitaxel aerosol concentration of 73.5 μg/L during the 63minute inhalation exposure was ˜11% lower from target average Paclitaxelaerosol concentration of 82.65 μg/L. The average exposure aerosolconcentration was within ±15% of target aerosol concentration which wasexpected for nebulized inhalation exposures. The particle sizedistribution was determined in terms of MMAD (GSD) for 6.0 mg/mLpaclitaxel particle formulation aerosols using cascade impactor as 2.0(2.2) μm. The recorded oxygen and temperature ranges were 19.7%-20.9%and 20.4° C.-20.8° C., respectively.

Paclitaxel deposited dose was calculated based on Paclitaxel averageaerosol concentration of 73.5 μg/L, average rodent body weight of 326 g,assumed deposition fraction of 10% and exposure duration of 63 minutes.The average achieved rodent deposited dose was determined to be 0.33mg/kg. The average achieved deposited dose was ˜11% lower when comparedto target deposited dose of 0.37 mg/kg due to expected variability (±15%from target).

All animals survived to their planned necropsy timepoint. At necropsy,several animals had minimal, red discolorations on the lungs. No otherabnormal gross observations were noted at necropsy. From body and lungweights obtained at necropsy, average terminal bodyweight (standarddeviation) was 346.26 g (24.01); and average lung weight (standarddeviation) was 1.60 g (0.13). Organ lung weights and lung weight to bodyweight ratios are common parameters used to assess potentialtoxicological responses to inhaled materials. Overall, the data indicatethat there was not a response with either of these endpoints.

Average paclitaxel concentration in plasma was 16.705 ng/mL at 0.5 hourspost exposure, then decreased gradually through the 24 hour timepointand was below the lower limit of quantification at all timepoints after24 hours. Average paclitaxel concentration in lung tissue was 21940 ng/gat 0.5 hours post exposure and decreased gradually to 419.6 ng/g by the168 hour timepoint. This indicates significant paclitaxel particleretention in the lung with minimal systemic exposure. The followingsampling timepoints are suggested for the PK study: 0.5 (±10 minutes), 6(±10 minutes), 12 (±10 minutes), 24 (±30 minutes), 48 (±30 minutes), 72(±30 minutes), 120 (±30 minutes) 168 (±30 minutes), 240 (±30 minutes)and 336 (±30 minutes) hours post exposure.

Example 2: Study FY17-008B—Paclitaxel Particle Aerosol InhalationExposure Study Executive Summary

The overall objective of this work was to conduct nose-only inhalationexposure to male rats with paclitaxel particle suspension formulationsof 6.0 mg/mL and 20.0 mg/mL. Rat inhalation exposures were conducted for65 minutes each.

Paclitaxel particle suspension formulations of 6.0 mg/mL and 20.0 mg/mLwere prepared as per instructions provided by the sponsor. Two Hospitakcompressed air jet nebulizers were used simultaneously at 20 psi foraerosolization of paclitaxel particle formulation into the rodentinhalation exposure chamber. During each exposure, aerosol concentrationwas measured from animal breathing zone by sampling onto 47-mm GF/Afilters at a flow rate of 1.0±0.5 L/minute. Particle size was determinedby sampling aerosols from animal breathing zone using Mercer stylecascade impactor at a flow rate of 2.0±0.1 L/minute. Filters wereanalyzed gravimetrically to determine total paclitaxel particle aerosolconcentration and via high performance liquid chromatography (HPLC) todetermine Paclitaxel aerosol concentration for each exposure. Oxygen andtemperature were monitored and recorded throughout the inhalationexposures.

The average total paclitaxel particle aerosol concentration andPaclitaxel aerosol concentration were determined to be 0.25 mg/L with aRSD of 7.43% and 85.64 μg/L with a RSD of 10.23%, respectively forinhalation exposures conducted with 6.0 mg/mL paclitaxel particleformulation. The measured average mass median aerodynamic diameter(geometric standard deviation) using cascade impactor was 1.8 (2.0) μmfor 6.0 mg/mL paclitaxel particle formulation aerosols. The averagetotal paclitaxel particle aerosol concentration and Paclitaxel aerosolconcentration were determined to be 0.46 mg/L with a RSD of 10.95% and262.27 μg/L with a RSD of 11.99%, respectively for inhalation exposuresconducted with 20.0 mg/mL paclitaxel particle formulation. The measuredaverage mass median aerodynamic diameter (geometric standard deviation)using cascade impactor was 2.3 (1.9) μm for 20.0 mg/mL paclitaxelparticle formulation aerosols.

The average Paclitaxel deposited dose of 0.38 mg/kg and 1.18 mg/kg werecalculated using equation 1 for a 65 minute exposure for 6.0 mg/mL and20.0 mg/mL paclitaxel particle formulation, respectively.

Formulation and Inhalation Exposure Formulation Preparation MaterialsTest Article

The test article used for inhalation exposure is shown below.

Paclitaxel Particles

Identity: Paclitaxel particles (sterile)Description: Novel dry powder formulation of Paclitaxel delivered as 306mg/vial

Supplier: US Biotest Manufacturer: CritiTech Storage Conditions: AmbientVehicle

The vehicles used for preparation of paclitaxel particle formulationsare shown below.

Polysorbate 80

Identity: Sterile 1% Polysorbate 80 in 0.9% sodium chloride forinjectionDescription: Clear liquid

Supplier: US Biotest Manufacturer: CritiTech Storage Conditions: AmbientNormal Saline

Identity: Sterile 0.9% sodium chloride for injection, USPDescription: Clear liquid

Manufacturer: Hospira, Inc, IL Storage Conditions: Ambient Formulationand Inhalation Exposure Formulation Preparation

Paclitaxel particle formulation of 6.0 mg/mL was prepared as follows:Briefly, 5.0 mL of 1% Polysorbate 80 was added to the vial containingpaclitaxel particles (306 mg). The vial was shaken vigorously andinverted to ensure wetting of all particles present in the vial.Immediately after shaking, 46 mL of 0.9% Sodium Chloride solution wasadded to the vial and vial was shaken for at least 1 minute to make suresufficient mixing and proper dispersion of suspension.

The paclitaxel particle formulation procedure described above for 6.0mg/mL formulation was used to prepare 20.0 mg/mL paclitaxel particleformulation with an exception of 10.3 mL of 0.9% sodium chloridesolution was added to the vial instead of 46 mL used for the 6.0 mg/mLformulation.

Resultant formulations were left undisturbed for at least 5 minutes toreduce any air/foam in the vial before placing it in nebulizer foraerosolization work. The final formulation of 6.0 mg/mL was kept at roomtemperature and nebulized within 2 hours after reconstitution. The finalformulation of 20.0 mg/mL was kept at room temperature and nebulizedwithin 30 minutes after reconstitution.

Experimental Design

Thirty (30) Sprague Dawley rats were exposed to a single “clinicalreference” dose of intravenous ABRAXANE® (paclitaxel: target dose 5.0mg/kg), thirty (30) Sprague Dawley rats were exposed to the paclitaxelparticle formulations disclosed herein (target dose of 0.37 mg/kg) andthirty (30) Sprague Dawley rats were expose to the paclitaxel particleformulations (target dose of 1.0 mg/kg) by nose only inhalation on asingle occasion. Three animals (n=3) were euthanatized at 0.5 (±10minutes), 6 (±10 minutes), 12 (±10 minutes), 24 (±30 minutes), 48 (±30minutes), 72 (±30 minutes), 120 (±30 minutes), 168 (±30 minutes), 240(±30 minutes), and 336 (±30 minutes) hours post exposure for blood(plasma) and lung tissue collections. Non-compartmental analyses wereperformed on plasma and lung tissue to identify duration of detectableamounts of paclitaxel post exposure for each dose group.

Exposure System Set-up/Aerosol Generation: As in example 1

Aerosol Concentration Monitoring: As in Example 1 Particle SizeDistribution: As in Example 1 Deposited Dose Calculation: As in Example1 Results Exposure Results Aerosol Concentration and Particle Size

Aerosol concentration was monitored throughout each paclitaxel particleformulation aerosol exposure using 47-mm GF/A filters from breathingzone of the animals on nose-only exposure chamber. Seven 47-mm GF/Afilters were sampled during each exposure. Filters FS-1 through FS-6were sampled for 10 minutes each and filter FS-7 was sampled for 5minutes during each low and high dose groups. Particle size was measuredusing Mercer style cascade impactor from animal breathing zone on theexposure chamber. Tables 3 and 4 show total and Paclitaxel aerosolconcentrations measured by sampling GF/A filters during low dose andhigh dose exposures, respectively.

TABLE 3 Aerosol concentrations during FY17-008B low dose inhalationexposure. Total Aerosol Paclitaxel Aerosol Filter ID Conc. (mg/L) Conc.(μg/L) FS-1-L 0.247 80.05 FS-2-L 0.242 81.79 FS-3-L 0.252 87.09 FS-4-L0.296 104.38 FS-5-L 0.247 78.47 FS-6-L 0.249 82.50 FS-7-L 0.244 85.19Average 0.25 85.64 SD 0.02 8.76 % RSD 7.43 10.23

TABLE 4 Aerosol concentrations during FY17-008B high dose inhalationexposure. Total Aerosol Paclitaxel Aerosol Filter ID Conc. (mg/L) Conc.(μg/L) FS-1-H 0.383 212.53 FS-2-H 0.412 239.28 FS-3-H 0.494 291.44FS-4-H 0.516 296.56 FS-5-H 0.456 254.67 FS-6-H 0.501 289.50 FS-7-H 0.431251.88 Average 0.46 262.27 SD 0.05 31.45 % RSD 10.95 11.99

The particle size (aerosol droplet size) distribution was determined interms of MMAD (Median of the distribution of airborne particle mass withrespect to the aerodynamic diameter) (GSD; accompanies the MMADmeasurement to characterize the variability of the particle sizedistribution) for each paclitaxel particle formulation aerosols usingcascade impactor. For 6.0 mg/mL and 20.0 mg/mL paclitaxel particleformulation aerosols the MMAD (GSD) were determined to be 1.8 (2.0) μmand 2.3 (1.9) μm, respectively. FIGS. 2 and 3 show particle sizedistribution for 6.0 mg/mL and 20.0 mg/mL paclitaxel particleformulations aerosols, respectively.

Deposited Dose

Paclitaxel deposited dose was calculated based on Paclitaxel averageaerosol concentration, average rat body weight, assumed depositionfraction of 10% and exposure duration of 65 minutes for each low doseand high dose paclitaxel particle formulation exposures by usingequation 1. Table 5 shows average Paclitaxel aerosol concentration,average rat body weight, exposure time and deposited dose for eachexposure. The average achieved rodent deposited dose was determined tobe 0.38 mg/kg and 1.18 mg/kg for 6.0 mg/kg and 20.0 mg/kg paclitaxelparticle formulation exposures, respectively.

TABLE 5 Paclitaxel deposited dose for low and high dose paclitaxelparticle formulation inhalation exposures. paclitaxel Paclitaxelparticles Avg. Exposure Deposited Dose Formulation Aerosol Avg. Rat TimeDose Level Conc. (mg/mL) Conc. (μg/L) Weight (g) (min.) (mg/kg) Low 6.085.64 420.4 65 0.38 High 20.0 262.27 420.5 65 1.18

Oxygen and Temperature

Oxygen and temperature were monitored throughout the paclitaxel particleformulation aerosols exposures. The recorded oxygen and temperatureranges were 19.8%-20.9% and 20.7° C.-20.8° C., respectively for 6.0mg/mL paclitaxel particle formulation exposure. For 20.0 mg/mLpaclitaxel particle formulation exposure, the recorded oxygen value was19.8% throughout the exposure and temperature range was 20.7° C.-20.8°C.

Preliminary data is shown in FIGS. 4-6.

Example 3 Evaluating Efficacy of Inhaled Paclitaxel ParticleFormulations in the Nude Rat Orthotopic Lung Cancer Model—Study FY17-095Executive Summary

One hundred twenty-seven (127) NIH-rnu Nude Rats were x-irradiated toinduce immunosuppression on Day −1. On Day 0 animals were dosed withCalu3 tumor cells by intratracheal (IT) instillation. Animals underwenta growth period of three weeks. During the third week, animals wererandomized by body weight stratification into 5 study groups. StartingWeek 4, animals in Group 2 received a once weekly dose of ABRAXANE® byintravenous (IV) dosing (5 mg/kg) on Days 22, 29 and 36. Animals inGroups 3 and 4 received once weekly (Monday) inhalation (INH) dose ofpaclitaxel particle formulations at low (0.5 mg/kg) and high (1.0 mg/kg)target doses, respectively. Animals in Groups 5 and 6 received a twiceweekly (Monday and Thursday) target inhalation dose of paclitaxelparticle formulations at low (0.50 mg/kg) and high (up to 1.0 mg/kg)doses respectively. Animals in Group 1 were left untreated as a controlof normal tumor cell growth. All animals were necropsied during Week 8.

All animals survived to their designated necropsy timepoint. Clinicalobservations related to the model included skin rash and laboredbreathing. All groups gained weight at about the same rate throughoutthe course of the study.

The inhalation exposure average Paclitaxel aerosol concentration foronce weekly Low Dose and twice weekly Low Dose paclitaxel particleformulation groups was 270.51 μg/L and 263.56 μg/L, respectively. Theinhalation exposure average Paclitaxel aerosol concentration for onceweekly High Dose and twice weekly High Dose paclitaxel particleformulation groups was 244.82 μg/L and 245.76 μg/L, respectively.

Doses were based on average aerosol paclitaxel concentration, mostrecent average group bodyweight, the assumed deposition fraction of 10%,and an exposure duration of 33 (Low-Dose) or 65 (High-Dose) minutes.During four weeks of treatment, the average achieved rodent depositeddose for the once weekly Low Dose paclitaxel particles formulation groupand twice weekly Low Dose paclitaxel particles formulation group were0.655 mg/kg and 0.640 mg/kg (1.28 mg/kg/week), respectively. The averageachieved rodent deposited dose for the once weekly High Dose paclitaxelparticles formulation group and twice weekly High Dose paclitaxelparticles formulation group were 1.166 mg/kg and 1.176 mg/kg (2.352mg/kg/week), respectively. For the group receiving IV injections ofABRAXANE®, the average dose on Day 22, 29 and 36 was 4.94, 4.64 and 4.46mg/kg respectively.

At scheduled necropsy, the majority of animals from each group had tannodules on the lungs and/or red or tan patchy discolorations of thelung. Other sporadic observations included an abdominal hernia in oneanimal and a nodule on the pericardium in another animal. No otherabnormal gross observations were noted at necropsy.

In the ABRAXANE® treated animal's lung weights, the lung to BW ratiosand lung to brain weight ratios were significantly lower compared toUntreated Controls. The once weekly paclitaxel particle formulation HighDose group had similar weights to the ABRAXANE® group and significantlylower lung weights and lung to brain ratios compared to UntreatedControls.

Histologically, lungs of the majority of animals in all groups containedsome evidence of tumor formation. Tumor formation was characterized bythe presence of expansile variably sized small masses randomly scatteredwithin the lung parenchyma and larger expanded and coalescing massesthat effaced up to 75% of the lung parenchyma, smaller airways and bloodvessels. The larger masses were distributed primarily in the hilarregions or juxtaposed at the axial airway and the smaller masses weregenerally located peripherally.

The primary morphologic cellular characteristics of the lung tumormasses varied from the presence of undifferentiated to a fairly welldifferentiated pattern of adenocarcinoma of the lung. The predominanttumor cell type showed an undifferentiated adenocarcinoma morphology;the cells were pleomorphic, large, anaplastic, pale amphophilic-stainingwith fine intracytoplasmic vacuoles resembling mucoid vesicles,exhibited moderate to marked anisokaryosis, and were observed to beindividualized or growing in sheets and lacking clear-cut featurestowards differentiation to adenocarcinoma. However, the cellularmorphologic characteristics that were observed within other masses orgrowing within the previously described undifferentiated masses weremore organized and consistent with well differentiated lungadenocarcinoma demonstrating clear acinar gland differentiation. Theseamphophilic staining tumor cells were primarily arranged in nests orglandular patterns which were observed to be bound by alveolar septae.Mitotic figures were rarely observed in this tumor cell population. Lessfrequently observed within these masses were focal areas ofprimitive-appearing relatively small Primitive Tumor Cells with small tomoderate amounts of pale basophilic staining cytoplasm, ovoid andvariably vesicular nuclei, and moderate anisokaryosis. These PrimitiveTumor Cells were observed to be growing randomly and in sheets.Increased numbers of mitotic figures and apoptotic bodies were notedmost often in this basophilic Primitive Tumor Cell population.Inflammation, characterized by mixed inflammatory cell (predominatelyeosinophils, lymphocytes, foamy macrophages and the occasional giantcell) infiltration accompanied by interstitial fibrosis was commonlyobserved. Significant parenchymal necrosis was uncommon to absent.

The pathologist considered the presence of scalloping of the edges ofthe individual tumor masses characterized by gradual loss of tumorcells, to complete loss of tumor cells with residual fibrosis connectivetissue scaffolding of the lung parenchyma and accompanied by invasion offoamy macrophages to be evidence of Tumor Regression.

Compared to the positive control Grp. 1 and the ABRAXANE® treatedcomparative Grp. 2, there was a decreased overall lung tumor burden inthe paclitaxel particle formulation treated groups (Grp. 3-6)characterized by a decrease in the severity of adenocarcinoma tumormasses and Primitive Tumor Cell population as well as evidence of TumorRegression. No other treatment-related lesions or findings wereobserved. Extensive mononuclear cell infiltration was observed in thelungs of animals receiving paclitaxel particle formulation throughinhalation. As the model used is T cell deficient, it is likely that thecells are B cells or NK cells. It is hypothesized that the localized,likely higher concentration exposure of the tumor to paclitaxelparticles affected the tumors leading to an alteration in theenvironment to draw the mononuclear cellular infiltrate into the lung.

Objectives

The objective of this study was to evaluate the efficacy of inhaledpaclitaxel particle formulation compared to a clinical reference dose ofintravenous administered ABRAXANE® in reducing tumor burden in anorthotopic model of lung cancer.

Materials and Methods Test System Species/Strain: NIH-rnu Nude Rats

Age of Animals at Study Start: 3-5 weeks old

Body Weight Range at Study Start: Approximately 150-200 g

Number on Study/Sex: 127 Males (120 study animals and 7 spares)

Source: Envigo

Identification: Permanent maker tail marking

ABRAXANE® Formulation

The clinical reference material used for IV formulation was the drugproduct ABRAXANE®. The drug product was reconstituted to 5.0 mg/mL withsaline on the day of dosing and was stored per manufacturer'sinstructions.

Paclitaxel Particle Formulation

The 20.0 mg/ml paclitaxel particle formulations for exposures wereprepared per the sponsor recommendations. Specifically, the paclitaxelparticles were reconstituted with 1% polysorbate 80. The vial was shakenby hand until all particles were wetted. Additional 0.9% sodium chloridefor injection was added (to the desired concentration target) and thevial was shaken by hand for another minute. Shaking continued until nolarge clumps were visible and the suspension was properly dispersed.

Resultant formulations were left undisturbed for at least 5 minutes toreduce any air/foam in the vial before placing it in a nebulizer foraerosolization work. The final formulation was kept at room temperatureand nebulized within 2 hours after reconstitution. The final 20.0 mg/mLwas kept at room temperature and nebulized within 30 (+5) minutes afterreconstitution.

Experimental Design

One hundred twenty-seven (127) animals were used for study. Prior tox-irradiation and dosing of tumor cells, 7 animals were designated asspares (spare animals did not have irradiations or cell lineinstillations). On Day −1 all study animals were x-irradiated to induceimmunosuppression. On Day 0 animals were dosed with Calu3 tumor cells byintratracheal (IT) instillation. Animals underwent a growth period ofthree weeks. During the third week, animals were randomized by bodyweight stratification into the groups outlined in Table 6 below.Starting Week 4, animals in Group 2 received a once weekly target doseof ABRAXANE® by intravenous (IV) dosing (5 mg/kg). Animals in Groups 3and 4 received once weekly (Monday) inhalation (INH) target dose ofpaclitaxel particle formulations at low (0.5 mg/kg) and high (1.0 mg/kg)doses, respectively. Animals in Groups 5 and 6 received a twice weekly(Monday and Thursday) inhalation target dose of paclitaxel particleformulations at low (0.50 mg/kg) and high (1.0 mg/kg) respectively.Animals in Group 1 were left untreated as a control of normal tumor cellgrowth. All animals were necropsied during Week 8.

TABLE 6 Experimental Design Group Cell Target Dose Treatment ExposureDescription N= Irradiation Line Route and Frequency* FormulationDuration Necropsy* 1 Control 20 Day −1 Calu 3, N/A N/A N/A N/A Week 8 2IV 20 IT IV up to 5 ABRAXANE ® N/A ABRAXANE ® instillation mg/kg** (5mg/ml) 3 paclitaxel 20 Day 0 INH 0.5 mg/kg, 20.0 mg/mL 33 min particleonce weekly paclitaxel formulation particle Low Once formulation Weekly(1×) 4 paclitaxel 20 INH 1.0 mg/kg, 20.0 mg/mL 65 min particle onceweekly paclitaxel formulation particle High Once formulation Weekly (1×)5 paclitaxel 20 INH 0.5 mg/kg, 20.0 mg/mL 33 min particle twice weeklypaclitaxel formulation particle Low- Twice formulation Weekly (2×) 6paclitaxel 20 INH 1.0 mg/kg, 20.0 mg/mL 65 min particle twice weeklypaclitaxel formulation particle High Twice formulation Weekly (2×)*Treatment occurred during Week 4-8. Necropsy occurred during Week 8.**ABRAXANE ® target dose: 5.0 mg/kg based on bodyweight; target dosevolume: not to exceed 250 μL, frequency: Day 1, 8, and 15 of each 21 daycycle beginning during Week 4.

Husbandry, Quarantine and Assignment to Study

After quarantine all animals were weighed and randomized to remove the 7spares based on body weights. From Week 1 to Week 3 animals wereidentified by cage cards (LC numbers) and tail markings.

During Week 3, prior to beginning treatment, animals were weighed andrandomized into the groups listed above by body weight stratificationand assigned a Study ID. From this point forward, animals wereidentified by cage cards and sharpie tail marking.

Immunosuppression and Irradiation

On Day −1, animals underwent whole body x-ray exposure with ˜500 rads(Phillips RT 250 X-ray Therapy Unit, Phillips Medical Systems, Shelton,Conn.) set at 250 kVp, 15 mA, and a source-to-object distance of 100 cm.The animals were placed in a pie chamber unit, 2-3 animals per slice ofpie. The irradiation process took ˜10-15 minutes.

Tumor Cell Implantation

On Day 0, animals received tumor cells (Calu3) administered by IT.Briefly, after being anesthetized by 3-5% isoflurane in an inductionchamber, the animal was placed with upper incisors hooked on an inclinedhanging instillation platform. The animals tongue was gently securedwhile the stylet is inserted just past the larynx and into the trachea.A volume of cells in EDTA suspension (target dose volume: 500 μL;concentration: approximately 20×10⁶ per 0.5 mL) was delivered to thelungs via intratracheal instillation. After the instillation, theanimals' breathing and movement was monitored carefully. Following tumorcell implantation, animals underwent a tumor growth period ofapproximately 3 weeks prior to treatment to allow for tumor cellengraftment and the development of lung cancer.

Calu3 Growth and Preparation

Calu3 cells were grown at 37° C. with 5% CO₂ in cell culture flasks.They were grown in Roswell Park Memorial Institute (RPMI) 1640 mediawith 10% fetal bovine serum (FBS) until 80% confluence. Cells weremaintained until the day of instillation. Prior to instillation theywere harvested by washing with PBS, then trypsin was added to removecells from the flask. The cells were neutralized with RPMI 1640 mediacontaining 10% FBS. They were then centrifuged at 100×g for 5 minutes;the media was removed and the cells were resuspended to a concentrationof 20 million cells in 450 μL of serum free RPMI. Prior to instillation,50 μL of 70 μM EDTA was added to the cell suspension for a total IT dosevolume of 500 μL per rat.

Body Weights and Daily Observations

Body weights were collected for randomization, weekly through Week 3,twice weekly beginning at Week 4 through the end of the study, and atnecropsy.

Each animal on study was observed twice daily for any clinical signs ofabnormality, morbidity or death. Technicians observed animals duringdosing and bodyweight sessions.

ABRAXANE® Administration IV-Tail Vein Injections

ABRAXANE® (5 mg/mL, maximum dose volume of 250 μL) was administered toanimals in Group 2 by IV tail vein injection on Days 22, 29 and 36.

Paclitaxel Particle Formulation Administration—Nose-Only AerosolExposures Conditioning

Animals were conditioned to nose-only exposure tubes for up to 70minutes. Three conditioning sessions occurred over three days prior toexposure, with the first session lasting 30 minutes, the second 60minutes and the third 70 minutes. They were monitored closely throughoutthe conditioning periods and during exposures to assure that they didnot experience more than momentary distress.

Exposure System

Aerosols were generated with two compressed air jet Hospitak nebulizersas shown in FIG. 7 at a nebulizer pressure of 20 psi. paclitaxelparticle suspension formulation of 20.0 mg/mL was used for low dose andhigh dose exposures. Aerosols were directed through a delivery line intoa 32-port nose-only exposure chamber. The rodent inhalation exposureswere conducted for 33 or 65 minutes. paclitaxel particle suspensionaerosol was generated with a set of two Hospitak compressed air jetnebulizers (used for up to 40 (±1) minutes), then replaced with a secondset of two Hospitak nebulizers for remaining exposure duration. Oxygenand temperature were monitored and recorded throughout each inhalationexposure

Concentration Monitoring

Aerosol concentration monitoring was conducted by collecting aerosolsonto pre-weighed GF/A 47-mm filters. The filters were sampled fromanimals breathing zones of the nose-only exposure chamber throughouteach inhalation exposure. The aerosol sampling flow rate through GF/Afilters was maintained at 1.0±0.5 L/minute. Filters were collectedthroughout each exposure duration every 10-minutes except for the lastfilter. With the low-dose exposures (groups 3 and 5) lasting 33 minutes,the final filter was collected after 13 minutes and with the high-doseexposures (groups 4 and 6) lasting 65 minutes, the final filter wascollected after 15 minutes. After sample collection filters were weighedto determine the total aerosol concentration in the exposure system.

Post weighing, each filter was placed in a 7 mL glass vial. The filtersin glass vials were extracted and analyzed by High Performance LiquidChromatography (HPLC) to quantify the amount of Paclitaxel collectedonto the filters. The total aerosol concentration and Paclitaxel aerosolconcentrations were calculated for each filter by dividing the totalamount of aerosols and Paclitaxel aerosols collected with total air flowthrough the filter. The average Paclitaxel aerosol concentration wasused to calculate the achieved average deposited dose of Paclitaxel tothe rodent lungs using Equation 1 as shown in the Determination of Dosesection below.

Determination of Dose

Deposited dose was calculated using Equation 1. In this calculation theaverage aerosol concentration measured from the exposures along withaverage group body weights for rats were used. In this manner theestimated amount of Paclitaxel deposited in the rat lungs was calculatedusing the measured Paclitaxel aerosol concentration.

$\begin{matrix}{{{DD}\left( {{\mu g}\text{/}{kg}} \right)} = \frac{A\; {C\left( {{\mu g}\text{/}L} \right)} \times {{RMV}\left( {L\text{/}{\min.}} \right)} \times {DF} \times {T\left( {\min.} \right)}}{{BW}({kg})}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

Where:

Deposited Dose=(DD) μg/kg

²Respiratory minute volume (RMV)=0.608×BW^(0.852)

Aerosol exposure concentration (AC)=Paclitaxel aerosol concentration(μg/L)

Deposition Fraction (DF)=assumed deposition fraction of 10%

BW=average body weight (at randomization; Day −1) of animals on study(kg)

Euthanasia and Necropsy

At scheduled necropsy, animals were euthanized by intraperitonealinjection of an overdose of a barbiturate-based sedative.

Blood and Tissue Collection

For all necropsies a terminal body weight and brain weight wascollected. For scheduled euthanasia blood (for plasma) was collected bycardiac puncture into a K₂EDTA tube. The lungs were removed and weighed.A section of lung tissue containing a tumor, a tracheobronchial lymphnode, was frozen in liquid nitrogen for potential future analysis. Theremaining lung was fixed for potential histopathology.

Histopathology

Fixed left lung lobes were trimmed in a “bread loaf” manner andalternate sections were placed in 2 cassettes to yield 2 slides eachwith 3 representative sections of the left lung. Tissues were processedroutinely, paraffin embedded, sectioned at ˜4 μm, mounted, and stainedwith hematoxylin and eosin (H&E) for microscopic examination. Findingswere graded subjectively, semi-quantitatively.

Sections of lung (1-4/animal) obtained from 60 out of the 120 treatednude rats on study, trimmed longitudinally, were processed to H & Estained glass slides for light microscopic evaluation.

During this review, the microscopic findings were recorded and thentransferred to an electronic pathology reporting system(PDS-Ascentos-1.2.0, V.1.2), which summarized the incidence andseventies of the lung burden characteristics data and tabulated theresults and generated the individual animal data. The lungs from the 60nude rats were examined histologically: Group 1 [1001-1010], Group 2[2001-2010], Group 3 [3001-3010], Group 4 [4001-4010], Group 5[5001-5010] and Group 6 [6001-6010]). In order to assess the level oftumor burden in these lungs, the lungs were evaluated and scored duringhistopathologic examination. For each cumulative lung burdencharacteristic diagnosis: 1) Adenocarcinoma (undifferentiated anddifferentiated), 2) Primitive Tumor Cells (poorly differentiatedpleomorphic cells) and 3) Tumor Regression, the lungs were gradedsemi-quantitatively using a 4-point grading scale indicating the percentinvolvement of the overall lung tissue provided as follows: 0=noevidence, 1=minimal (˜1-25% total area of lung sections involved),2=mild (˜25-50% total area of lung sections involved), 3=moderate(˜50-75% total area of lung sections involved), and 4=marked (˜75-100%total area of lung sections involved).

HistoMorphometry

Histomorphometric analyses was performed using fixed left lung lobes ofthe first 10 animals from each group. Tissue was trimmed using amorphometry (“bread slice”) style trim. Briefly, trimming started at arandom point between 2 and 4 mm from the cranial end of the lung. Eachlung section was cut approximately 4 mm thick. Odd numbered sectionswere placed caudal side down in cassette 1 while even numbered sectionswere placed in cassette 2. Tissue sections were then processed, paraffinembedded, and sectioned at 4 μm and stained with hematoxylin and eosin(HE) for examination. Both slides (odd and even slices) were used todetermine an average tumor fraction per animal.

Morphometric analysis was performed on the hematoxylin and eosin (HE)stained lung tissue from the designated animals by Lovelace Biomedical.Whole slides (2 per animal containing transverse sections of the entireleft lung) were scanned using a Hamamatsu Nanozoomer. Scans wereanalyzed with Visiopharm Integrator System software (VIS, version2017.2.5.3857). Statistical analysis of tumor area fraction wasperformed in GraphPad Prism 5 (version 5.04).

Computerized image quantification designed to quantify the amount oftumor area present on each slide was performed on all left lung tissueusing the whole slide scans. The Visiopharm Application for quantifyingthe area of lung metastases was used to differentiate tumor cells fromnormal lung tissue based on cell density, staining intensity, and sizeand staining intensity. It is noted that this quantitation based uponsimple H&E staining will not be perfect (i.e. it is not capable of fullydiscriminating between types of tumor tissue, necrotic and viable tumortissue, and some normal structures may be included as tumor). The valuein application of this process to H&E sections is that it is an unbiasedapproach to tumor quantification. The area of the whole piece of lung isdetermined, and the area occupied by structures identified as metastasesis then expressed as a percentage of the total area. Minor adjustment ofthe area to be analyzed to ensure extrapulmonary structures are excludedand the entire lung is included may be performed manually. Other manualmanipulations are avoided in order to ensure consistency across allgroups and remove potential for introduction of bias. If possible,development of specific immunohistochemical stains to identify onlytumor tissue would increase specificity of this analysis.

Blood Collection and Processing

Blood collected at necropsy was processed to plasma by centrifugation ata minimum of 1300 g at 4° C. for 10 minutes. Plasma samples were storedat −70 to −90° C. until analysis or shipment to sponsor.

Results Clinical Observation, Survival, and Bodyweights

All animals survived to their designated necropsy timepoint. Clinicalobservations related to the model included skin rash and laboredbreathing. One animal was observed to have an upper abdominal hernia.Per vet recommendation the animal was switched with a Group 1 (UntreatedControl) that would not undergo inhalation exposures therefore noexposure tube restraint would be necessary.

FIG. 8 shows the average body weights through the duration of the study.FIG. 9 shows the percent change in average body weights from Day 0. Allgroups gained weight at about the same rate through the course of thestudy.

ABRAXANE® IV Tail Vein Injections

For the group receiving IV injections of ABRAXANE®, the average dose onDay 22, 29 and 36 was 4.94, 4.64 and 4.46 mg/kg respectively.

Paclitaxel Particle Exposures Aerosol Concentrations and Deposited Dose

Total aerosol and Paclitaxel aerosol concentrations were measured bysampling of GF/A filters during each exposure. The inhalation exposureaverage Paclitaxel aerosol concentration for once weekly Low Dose andtwice weekly Low Dose paclitaxel particle formulation groups were of270.51 μg/L and 263.56 μg/L, respectively. The inhalation exposureaverage Paclitaxel aerosol concentration for once weekly High Dose andtwice weekly High Dose paclitaxel particle formulation groups were of244.82 μg/L and 245.76 μg/L, respectively. The oxygen and temperaturelevels were monitored throughout each exposure.

Doses were based on average aerosol paclitaxel concentration, mostrecent average group bodyweight, the assumed deposition fraction of 10%and an exposure duration of 33 or 65 minutes. During four weeks oftreatment, the average achieved rodent deposited dose for the onceweekly Low Dose paclitaxel particle formulation group and twice weeklyLow Dose paclitaxel particle formulation group were 0.655 mg/kg and0.640 mg/kg (1.28 mg/kg/week), respectively.

The average achieved rodent deposited dose for the once weekly High Dosepaclitaxel particle formulation group and twice weekly High Dosepaclitaxel particle formulation group were 1.166 mg/kg and 1.176 mg/kg(2.352 mg/kg/week), respectively.

Particle Size (MMAD & GSD)

The particle size distribution was determined in terms of Mass MedianAerodynamic Diameter (MMAD) and Geometric Standard Deviation (GSD) foreach paclitaxel particle formulation aerosols using cascade impactor.For the 20.0 mg/mL paclitaxel particle formulation aerosols the averageMMAD was determined to be 2.01 μm and a GSD of 1.87.

Necropsy Observations and Organ Weights

All animals survived to their designated necropsy timepoint. At necropsyanimals from each group had tan nodules on the lungs and/or red or tanpatchy discolorations of the lung. Other sporadic observations includedan abdominal hernia in one animal and a nodule on the pericardium inanother animal. No other abnormal gross observations were noted atnecropsy. One animal did not have any visible tumors (nodules) at thetime of necropsy.

Individual animal organ weight data is shown graphically in FIG. 10,FIG. 11 and FIG. 12. In ABRAXANE® treated animal's lung weights, lung toBW ratios and lung to brain weight ratios were significantly lowercompared to Untreated Controls. The once weekly paclitaxel particleformulation High Dose group had similar weights to the ABRAXANE® groupand significantly lower lung weights and lung to brain ratios comparedto Untreated Controls. The once weekly Low Dose, paclitaxel particleformulation twice weekly Low Dose and twice weekly High Dose paclitaxelparticle formulation groups generally had similar average lung weightsand ratios.

Morphometry

All treatment groups showed a decrease in average lung tumor fractionwhen compared to the control group; however, there was no statisticallysignificant difference between groups. There was also no statisticallysignificant difference between IV ABRAXANE® treatment and any of thepaclitaxel particle formulation treatment regimens in regards to thetumor area fraction examined on cross sectional lung slides. As istypical of this model, there is wide variability between animals withinall groups in the tumor fraction. These data should be considered incombination with other indicators of lung tumor burden in this modelincluding lung to brain weight ratios and standard histopathology forfinal interpretation. It is important to note that morphometric analysisand histopathologic examination was performed on fixed lung tissue fromthe left lobe while other analyses on lung tissue may be performed onfrozen tissue from the right lung lobes. Average tumor area is shown inFIG. 13 and FIG. 14.

Pathology Results

As a result of the slide examination of the identified populations ofneoplastic cells the pathologist determined: (1) There was a slightdecrease in severity of an overall lung tumor burden of Adenocarcinoma(undifferentiated and differentiated cells) in all treated groups (Grp.2 (1.7), Grp, 3 (1.8), Grp. 4 (1.7), Grp 5 (1.6) and Grp. 6 (1.6)compared to the untreated Control Grp. 1 (2.1). (2) There was reductionin the Primitive Tumor Cell population evident by a decrease in theseverity in Grp. 3 (0.3), Grp. 4 (0.3), Grp 5 (0.2) and Grp 6 (0.2)compared to the corresponding control Grp 1 (0.9) and Grp 2 (1.0), and3) There was Tumor Regression present in Grp 3 (0.6), Grp 4 (1.0), Grp 5(0.8) and Grp 6 (1.0) compared to the corresponding control Grp 1 (0.0)and Grp 2 (0.1). The incidence and seventies of the lung burdencharacteristics data are summarized in Table 7, and in FIG. 15.Photomicrographs of the slides are shown in FIGS. 16 to 50.

TABLE 7 Incidences and Severities of Cumulative Lung Burden Table Groups1 2 IV 3 4 5 6 Control Abraxane Low 1x High 1x Low 2x High 2x AnimalNos. 1001- 2001- 3001- 4001- 5001- 6001- 1010 2010 3010 4010 5010 6010LUNG (NO. (10)  (10)  (10)  (10)  (10)  (10)  EX) Adeno- 10  10  10  910  10  carcinoma Minimal   2^(a) 4 5 3 5 5 Mild 5 5 2 4 4 3 Moderate 31 3 2 1 2 Marked  0^(b) 0 0 0 0 0 Average   2.1   1.7   1.8   1.7   1.6  1.7 Severity Grade Primitive 9 10  2 3 2 2 Tumor Cells Minimal 9 10  13 2 2 Mild 0 0 1 0 0 0 Moderate 0 0 0 0 0 0 Marked 0 0 0 0 0 0 Average  0.9   1.0   0.3   0.3   0.2   0.2 Severity Grade Tumor 0 1 6 5 6 5Regression Minimal 0 1 6 3 5 2 Mild 0 0 0 0 0 2 Moderate 0 0 0 1 1 0Marked 0 0 0 1 0 1 Average 0   0.1   0.6   1.0   0.8   1.0 SeverityGrade ^(a)Severity Grade based on a 4 point grading scale of 1 to 4: 1 =minimal, 2 = mild, 3 = moderate, 4 = marked ^(b)The presence ofa (0)indicates that there is no evidence histopathologically of the lesion inquestion

Histological Overview of Photomicrographs in FIGS. 16 to 50 GeneralObservations:

Control: Extensive levels of viable tumor with proliferating cells andlittle to no immune cell infiltration.ABRAXANE® IV: Many viable appearing tumor masses with some lymphocyticresponse along with some tumor regression.paclitaxel particle formulation 1× per week, High: Clearance of tumorfrom the lung with few viable tumor cells remaining. Masses remainingappear to be immune cell infiltrate and fibrosis.paclitaxel particle formulation 2× per week, Low: Some remaining tumornodules surrounded by immune cell infiltrate including macrophages andmononuclear cells.paclitaxel particle formulation 2× per week, High: Few tumor noduleswith immune infiltrate and stromal fibrosis replacing tumor.

Extensive mononuclear tumoricidal cell infiltration was observed in thelungs of animals receiving paclitaxel particle formulation throughinhalation. As the model used is T cell deficient, it is likely that thecells are B cells or NK cells, or both. B cells are responsible for theproduction of antibodies and can be involved in tumor cell killingthrough antibody-dependent cell mediated cytotoxicity (the antibodiesbind to cells expressing Fc Receptors and enhance the killing ability ofthese cells). NK cells are innate lymphoid cells that are crucial in thekilling of tumor cells. In patients with tumors, NK cell activity isreduced allowing for the growth of the tumor. Along with T cells, NKcells are the target of some check point inhibitors to increase theiractivity.

By the use of a wide array of surface receptors capable of deliveringeither triggering or inhibitory signals, NK cells can monitor cellswithin their environment to ascertain if the cell is abnormal (tumor orvirally infected) and should be eliminated through cytotoxicity.

The cytotoxicity and chemotaxis of NK cells can be modified by manypathological processes including tumor cells and their byproducts. Inresponse to certain signals their functions are enhanced or potentiated.In response to several Pathogen Associated Molecular Patterns (PAMPs) byusing different Toll Like Receptors (TLR); NK cells can increasecytokine production and/or cytolytic activity. Cytokines, includingIL-2, IL-15, IL-12, IL-18, and IFNs α/β can also modify the activity ofNK cells. NK cells are not simple cells that are only cytolyticeffectors capable of killing different tumor cell targets; rather, theyrepresent a heterogeneous population which can finely tune theiractivity in variable environmental contexts.

The tumor burden seems to be significantly reduced in the lungs of theanimals treated with paclitaxel particle formulation and is lower thanthat for ABRAXANE® IV. Therefore, the localized administration ofpaclitaxel in the form of paclitaxel particle formulation providesadditional potency. This is likely due to both the longer exposure tothe chemotherapy over time and the vigorous cellular infiltration to thesite of the tumor. This latter response appeared to be dependent on thedose density (actual dose and dose frequency).

Observations of Specific Photomicrographs:

FIG. 16: Subject 1006 (Control) Adenocarcinoma-3, Primitive-1,Regression-0. Low-power magnification (2×) showing the generaldistribution of undifferentiated, pleomorphic, large, anaplastic tumorcells within alveolar spaces or lining the alveolar septae. The majorityof cells do not have features of adenocarcinoma and appear in sheets ofcontiguous tumor. Many cells have basophilic staining cytoplasm, whileothers are large, anaplastic and contain pale amphophilic-staining. Notethe presence of a pre-existing resident population of alveolarmacrophages and the absence of tumor regression.FIG. 30: Subject 2003 (IV ABRAXANE®) Adenocarcinoma-1, Primitive-1,Regression-1. Low-power magnification (4×) showing the generaldistribution of tumor masses predominantly at the periphery as well asmultiple smaller expansive tumor masses filling alveolar spaces. Thetumor cells are pleomorphic, large, anaplastic and have paleamphophilic-staining, varying from undifferentiated to differentiatedpatterns of adenocarcinoma. Evidence of tumor regression is presentaround the periphery of the mass and primarily characterized by theinfiltration of macrophages.FIG. 36: Subject 2010 (IV ABRAXANE®) Adenocarcinoma-3, Primitive-1,Regression-0. Low-power magnification (2×) showing the generaldistribution of large expansive tumor mass filling most alveolar spacesas well as neoplastic cells in the periphery. Most tumor cells arepredominantly undifferentiated, pleomorphic, large, anaplastic with paleamphophilic-staining. The primitive cells are smaller, ovoid, and havemore basophilic staining cytoplasm with variable, vesicular nuclei andmoderate to marked anisokaryosis. Inflammatory cell infiltration arepredominantly neutrophils and macrophages. This image demonstrates anabsence of tumor regression.FIG. 39: Subject 4009 (IH paclitaxel particle formulation 1×/wk High)Adenocarcinoma-0, Primitive-0, Regression-4. Low-power magnification(2×) showing the general distribution of previously populated tumormasses, the presence of multiple small areas of fibrous connectivetissue, central collagenous stroma and fibrocytes are seen at theperipheral alveolar spaces as well as thickened alveolar septae supportsevidence of tumor regression. In addition, the alveolar spaces arecommonly filled with infiltrate of macrophages and lymphocytes togetherwith additional evidence of tumor regression.FIG. 42: Subject 5010 (IH paclitaxel particle formulation 2×/wk Low)Adenocarcinoma-1, Primitive-0, Regression-3. Low-power magnification(2×) showing the general distribution of previously populated tumormasses. Regressing masses are variably small and randomly distributed.Fibrous connective tissue is seen filling/replacing alveolar spaces andsuggests foci of regressing adenocarcinoma. Acute necrosis, fibrousconnective scaffolding, mixed cell infiltration of macrophages, giantcells and lymphocytes in the epithelium as well as around the stroma aresigns of tumor regression.FIG. 46: Subject 6005 (IH paclitaxel particle formulation 2×/wk High)Adenocarcinoma-1, Primitive-0, Regression-4. Low-power magnification(2×) showing the general distribution of previously populated tumormasses in multiple small areas of fibrous connective tissuefilling/replacing the alveolar spaces suggesting foci of previousinfiltrates of adenocarcinoma cells. Tumor regression is evidenced byfibrosis of previously populated tumor masses, central collagenousstromal core and fibrous connective tissue at the peripheryfilling/replacing the alveolar spaces, thickening of the septae as wellas the presence of fibrocytes filling the alveolar space infiltrated bylymphocytes and macrophages.

Conclusions

One hundred twenty-seven (127) NIH-rnu Nude Rats were x-irradiated toinduce immunosuppression on Day −1. On Day 0 animals were dosed withCalu3 tumor cells by intratracheal (IT) instillation. Animals underwenta growth period of three weeks. During the third week, animals wererandomized by body weight stratification into the groups. Starting Week4, animals in Group 2 received a once weekly dose of ABRAXANE® byintravenous (IV) dosing (5 mg/kg) on Days 22, 29 and 36. Animals inGroups 3 and 4 received once weekly (Monday) inhalation (INH) dose ofpaclitaxel particle formulation at low (0.5 mg/kg) and high (1.0 mg/kg)target doses, respectively. Animals in Groups 5 and 6 received a twiceweekly (Monday and Thursday) target inhalation dose of paclitaxelparticle formulation at low (0.50 mg/kg) and high (1.0 mg/kg) dosesrespectively. Animals in Group 1 were left untreated as a control ofnormal tumor cell growth. All animals were necropsied during Week 8.

All animals survived to their designated necropsy timepoint. Clinicalobservations related to the model included skin rash, labored breathing.All groups gained weight at about the same rate through the course ofthe study.

The inhalation exposure average Paclitaxel aerosol concentration foronce weekly Low Dose and twice weekly Low Dose paclitaxel particleformulation groups was 270.51 μg/L and 263.56 μg/L, respectively. Theinhalation exposure average Paclitaxel aerosol concentration for onceweekly High Dose and twice weekly High Dose paclitaxel particleformulation groups was 244.82 μg/L and 245.76 μg/L, respectively.

Doses were based on average aerosol paclitaxel concentration, mostrecent average group bodyweight, assumed deposition fraction of 10% andexposure duration of 33 or 65 minutes. During four weeks of treatment,the average achieved rodent deposited dose for the once weekly Low Dosepaclitaxel particle formulation group and twice weekly Low Dosepaclitaxel particle formulation group were 0.655 mg/kg and 0.640 mg/kg(1.28 mg/kg/week), respectively. The average achieved rodent depositeddose for the once weekly High Dose paclitaxel particle formulation groupand twice weekly High Dose paclitaxel particle formulation group were1.166 mg/kg and 1.176 mg/kg (2.352 mg/kg/week), respectively. For thegroup receiving IV injections of ABRAXANE®, the average dose on Day 22,29 and 36 was 4.94, 4.64 and 4.46 mg/kg respectively.

At scheduled necropsy, the majority of animals from each group had tannodules on the lungs and/or red or tan patchy discolorations of thelung. Other sporadic observations included an abdominal hernia in oneanimal and nodule on the pericardium of another animal. No otherabnormal gross observations were noted at necropsy.

In ABRAXANE® treated animals, lung weights, lung to BW ratios and lungto brain weight ratios were significantly lower compared to UntreatedControls. The once weekly paclitaxel particle formulation High Dosegroup had similar weights to the ABRAXANE® group and significantly lowerlung weights and lung to brain ratios compared to Untreated Controls.

Compared to the positive control Grp. 1 and the ABRAXANE® treatedcomparative Grp. 2, there was a therapeutic effect as measured by lowerlung/brain weight ratio and lower overall lung tumor burden withoutapparent adverse events. Histological analysis of lung tumor burdentreated with inhaled paclitaxel particle formulation showed a decreasein tumor mass, a decrease in primitive tumor cell population, and anincrease in tumor regression. Extensive mononuclear cell infiltrationwas observed in the lungs of animals receiving paclitaxel particleformulation through inhalation. As the model used is T cell deficient,it is likely that the cells are B cells or NK cells. It is hypothesizedthat the localized, likely higher concentration exposure of the tumor topaclitaxel particles affected the tumors leading to an alteration in theenvironment to draw the mononuclear cellular infiltrate into the lung.

Example 4: Study FY 17-008B—Paclitaxel Particle Pharmacokinetic StudyExecutive Summary

Ninety (90) male Sprague Dawley rats were exposed to “clinicalreference” dose of paclitaxel, ABRAXANE® (paclitaxel protein boundparticles for injectable suspension, aka nab-paclitaxel), by intravenous(IV) bolus injection or paclitaxel particle formulation (target dose of0.37 or 1.0 mg/kg) by nose only inhalation on a single occasion. Threeanimals (n=3) were euthanatized at ten (10) timepoints from 0.5 to 336hours post exposure for blood (plasma) and lung tissue collections.Non-compartmental analysis (NCA) was performed on plasma and lung tissueto identify the duration of detectable amounts of paclitaxel postexposure for each dose group. Animals designated to the 336 hour timepoint from all groups had right lungs collected for liquidchromatography-mass spectrometry (LCMS) analysis while the left lungswere perfused with 10% neutral buffered formalin (NBF) and retained forpotential histopathology. In order to enable comparative histopathology,three spare animals (Naive Controls) were euthanized at the 336 hourtimepoint and lung collections were performed in the same manner.Animals designated to all other timepoints had all lungs individuallyfrozen for LCMS analysis.

The inhalation exposure average Paclitaxel aerosol concentration for LowDose and High Dose paclitaxel particle formulation groups was of 85.64μg/L and 262.27 μg/L, respectively. The average exposure aerosolconcentration was within ±15% of target aerosol concentration which wasexpected for nebulized inhalation exposures. The particle sizedistribution was determined in terms of MMAD (GSD) for each paclitaxelparticle formulation aerosols using a cascade impactor. For 6.0 mg/mLand 20.0 mg/mL paclitaxel particle formulation aerosols the MMAD (GSD)were determined to be 1.8 (2.0) μm and 2.3 (1.9) μm, respectively.

Paclitaxel deposited low-dose was calculated based on Paclitaxel averageaerosol concentration of 85.64 μg/L, average Day 0 group bodyweight of420.4 g, assumed deposition fraction of 10% and exposure duration of 65minutes; the average achieved rodent deposited dose was determined to be0.38 mg/kg for the Low Dose paclitaxel particle formulation group. Forthe High Dose paclitaxel particle formulation group, paclitaxel averageaerosol concentration of 262.27 μg/L, average Day 0 group bodyweight of420.5 g, assumed deposition fraction of 10% and exposure duration of 65minutes; the average achieved rodent deposited dose was determined to be1.18 mg/kg. The recorded oxygen and temperature ranges were 19.8%-20.9%and 20.7° C.-20.8° C., respectively for 6.0 mg/mL paclitaxel particleformulation exposure. For 20.0 mg/mL paclitaxel particle formulationexposure, the recorded oxygen value was 19.8% throughout the exposureand temperature range was 20.7° C.-20.8° C.

For the group receiving IV injections of ABRAXANE®, Day 1 bodyweightsranged from 386.1 to 472.8 g, this resulted in ABRAXANE® doses of2.6-3.2 mg/kg, with the average group dose being 2.9 mg/kg.

All groups gained weight through the course of the study. No abnormalclinical observations were noted through the duration of the study. Allanimals survived to their designated necropsy timepoint. All animalswere euthanized within the window intended for each time point.

At necropsy, approximately half of the animals from each group hadminimal to mild, tan discolorations on the lungs. Such observations areoften associated with inhalation exposures. Other transient observationsincluded an enlarged heart (animal #2016) and enlarged tracheobronchiallymph nodes. No other abnormal gross observations were noted atnecropsy. Histopathology showed lung and trachea from test and referencearticle treated rats were within normal limits and indistinguishablefrom those of na'ive rats under the conditions of this study. At the 336hour post-dosing sacrifice, macrophage accumulation which is common ininhalation studies as a physiologically normal response to exogenousmaterial deposited in the lung was not apparent within the lung sectionsof treatment animals examined for this study.

The NCA was designed to quantify the exposure (area under theconcentration versus time curve [AUC]), time to maximum concentration(T_(max)), maximum concentration (C_(max)) and when possible apparentterminal half-life (T_(1/2)).

The hypothesis for the novel paclitaxel particle formulation was thatthe formulation would result in increased retention of paclitaxel withinthe lung tissue and reduce the systemic exposure. The half-life withinsystemic plasma was unchanged for the formulation/doses tested and thehalf-life within the lung tissue was increased with the paclitaxelparticle formulation delivered by inhalation. The exposure to the lungtissue (dose normalized AUC) was increased when delivered as thepaclitaxel particle formulation by inhalation.

Collectively the data indicate a significant retention of paclitaxelparticles within the lung tissue when delivered via inhalation comparedto the IV “clinical reference”.

Objectives

The objective of this study was to determine the pharmacokinetics of thepaclitaxel particle formulation compared to a clinical reference dose ofpaclitaxel. The pilot pharmacokinetic (PK) data from Lovelace Biomedicalstudy FY 17-008A (Example 1 above) with paclitaxel particle formulationdosed by inhalation indicated a retention time beyond 168 hours in lungtissue. In this study, animals dosed with either a single low or highdose nose-only inhalation paclitaxel particle formulation or singleclinical reference dose of paclitaxel via intravenous (IV) tailinjection had plasma and lung tissue evaluated at timepoints from 0.5 to336 hours.

Materials and Methods Test System Species/Strain: Sprague Dawley Rats

Age of Animals at Study Start: 8-10 weeks of age

Body Weight Range at Study Start: 345-447 g

Number on Study/Sex: 95 Males (90 study animals and 5 spares)

Source: Charles River Laboratories (Kingston, N.Y.)

Identification: Permanent maker tail marking

ABRAXANE® Formulation

The clinical reference material used IV formulation was the drug productABRAXANE® (Manufacturer: Celgene Corporation, Summit, N.J.; Lot:6111880). The drug product was reconstituted to 5.0 mg/mL with saline(Manufacturer: Baxter Healthcare, Deerfield, 11.; Lot: P357889) on theday of dosing and was stored per manufacturer's instructions.

Paclitaxel Particle Formulation

The 6.0 mg/ml paclitaxel particle formulation for Low Dose groupexposures and 20.0 mg/ml paclitaxel particle formulation for High Dosegroup exposures were prepared per the sponsor recommendations.Specifically, the paclitaxel particles were reconstituted with 1%polysorbate 80. The vial was shaken by hand until all particles werewetted. Additional 0.9% sodium chloride for injection was added (to thedesired concentration target) and the vial was shaken by hand foranother minute.

Shaking continued until no large clumps were visible and the suspensionwas properly dispersed. Resultant formulations were left undisturbed forat least 5 minutes to reduce any air/foam in the vial before placing itin a nebulizer for aerosolization work. The final formulation of 6.0mg/mL was kept at room temperature and nebulized within 2 hours afterreconstitution. The final formulation of 20.0 mg/mL was kept at roomtemperature and nebulized within 30 minutes after reconstitution.

Experimental Design

Animals in Group 1 shown in Table 8 received a single “clinicalreference” dose (formulation concentration: 5 mg/mL, target dose: 5.0mg/kg based on bodyweight; target dose volume: not to exceed 250 μL) ofABRAXANE® (paclitaxel protein bound particles for injectable suspension)by IV tail vein injection. Animals in Group 2 and 3 in Table 9 wereexposed to paclitaxel particle formulation aerosols (target dose of 0.37or 1.0 mg/kg) by nose only inhalation (INH) on a single occasion per thestudy design below. Three animals (n=3) were euthanized at 0.5 (±10minutes), 6 (±10 minutes), 12 (±10 minutes), 24 (±30 minutes), 48 (±30minutes), 72 (±30 minutes), 120 (±30 minutes), 168 (±30 minutes) 240(±30 minutes) and 336 (±30 minutes) hours post exposure for blood(plasma) and lung tissue collections. Non-compartmental analyses wereperformed on plasma and lung tissue to identify duration of detectableamounts of paclitaxel post exposure for each dose group. Animalsdesignated to the 336 hour time point from all groups had right lungsindividually frozen for LCMS analysis while the left lungs were perfusedwith 10% neutral buffered formalin (NBF) and retained for potentialhistopathology. In order to enable comparative histopathology, threespare animals (Naive Controls) were also be euthanized alongside the 336hour timepoint and had have lung collections performed in the samemanner.

TABLE 8 Experimental Design Target Exposure PK timepoints Group N =Target Dose Route Duration (hours post exposure) 1 ABRAXANE ® 30 Up to5.0 mg/kg^(B) IV n/a N = 3 from each group “Clinical Reference” at 0.5,6, 12, 24, 48, 72, Dose 120, 168, 240 and 336^(A) 2 paclitaxel particle30 0.37 mg/kg INH up to 65 min hours post exposure formulation Low Dose3 paclitaxel particle 30  1.0 mg/kg INH up to 65 min formulation HighDose ^(A)Study animals from each group and three spares will have tissuecollections for LCMS analysis as well as potential histopathology at 336hours post exposure. ^(B)ABRAXANE ® (concentration: 5 mg/ml, targetdose: up to 5.0 mg/kg based on bodyweight with dose volume not to exceed250 μL) was administered to animals in Group I by IV tail vein injection

Husbandry, Quarantine and Assignment to Study

Male Sprague Dawley rats (6-8 weeks old) were obtained from CharlesRiver Laboratories (Kingston, N.Y.) and quarantined for 14 days. At theend of quarantine, animals were weighed and then randomized by weightfor assignment to study. Animals were identified by tail marking andcage card. Water, lighting, humidity, and temperature control weremaintained and monitored according to appropriate SOPs. Rats were fed astandard rodent diet ad libitum during non-exposure hours.

Body Weights and Daily Observations

Body weights were collected at randomization, daily throughout the studyand at euthanasia. Each animal on study was observed twice daily byComparative Medicine Animal Resources (CMAR) personnel for any clinicalsigns of abnormality, moribundity or death.

ABRAXANE® Administration IV—Tail Vein Injections

ABRAXANE® (concentration: 5 mg/mL, target dose: 5.0 mg/kg based onbodyweight; dose volume: not to exceed 250 μL) was administered toanimals in Group 1 by IV tail vein injection on a single occasion.

Paclitaxel Particle Administration—Nose-Only Aerosol Exposures

Conditioning

Animals were conditioned to nose-only exposure tubes for up to 70minutes. Three conditioning sessions occurred over three days prior toexposure, with the first session lasting 30 minutes, the second 60minutes and the third 70 minutes. They were monitored closely throughoutthe conditioning periods and during exposures to assure that they didnot experience more than momentary distress.

Exposure System

Aerosols were generated with two compressed air jet Hospitak nebulizersas shown in FIG. 7 above (see Example 3) at a nebulizer pressure of 20psi. paclitaxel particle suspension formulations of 6.0 mg/mL and 20.0mg/mL were used for low dose and high dose exposures, respectively. Bothformulations were aerosolized separately and aerosols were directedthrough delivery line into a 32-port nose-only exposure chamber. Therodent inhalation exposures were conducted each for 65 minutes.paclitaxel particle suspension aerosol was generated with a set of twoHospitak compressed airjet nebulizers (used for up to 40 (±1) minutes),then replaced with a second set of two Hospitak nebulizers for remainingexposure duration. Oxygen and temperature were monitored and recordedthroughout each inhalation exposure.

Concentration Monitoring

Same as in Example 1

Particle Size Determination

Same as in Example 1

Determination of Dose

Same as in Example 1

Euthanasia and Necropsy

Animals were euthanized at the time points in the study designs above byan intraperitoneal (IP) injection of euthanasia solution.

For 336 Hour Timepoint (and Spare Animals, n=3):

During necropsy, blood (for plasma) was collected by cardiac punctureinto a K₂EDTA tube. A whole lung weight was collected, the left lung wastied off and filled with neutral buffered formalin and saved forpotential histopathology. Right lung lobes were individually weighed andsnap frozen in liquid nitrogen and stored at −70 to −90° C. forbioanalytical analyses. Additionally, a full gross examination wasperformed by qualified necropsy personnel. External surfaces of thebody, orifices, and the contents of the cranial, thoracic, and abdominalcavities were examined. Lesions were described and recorded using a setof glossary terms for morphology, quantity, shape, color, consistency,and severity.

For all Other Timepoints:

During necropsy, blood (for plasma) was collected by cardiac punctureinto a K₂EDTA tube. A whole lung weight was collected, lung lobes wereindividually weighed and snap frozen in liquid nitrogen and stored at−70 to −90° C. for bioanalytical analyses. Additionally, a full grossexamination was performed by qualified necropsy personnel. Externalsurfaces of the body, orifices, and the contents of the cranial,thoracic, and abdominal cavities were examined. Lesions were describedand recorded using a set of glossary terms for morphology, quantity,shape, color, consistency, and severity.

Histopathology

Available fixed tissues were trimmed. Fixed left lung lobes were trimmedto yield a typical toxicologic pathology style section with airways.Tissues were processed routinely, paraffin embedded, sectioned at ˜4 μm,mounted, and stained with hematoxylin and eosin (H&E) for microscopicexamination. Findings were graded subjectively, semi-quantitatively by asingle pathologist experienced in toxicologic pathology on a scale of1-5 (1=minimal, 2=mild, 3=moderate, 4=marked, 5=severe). The Provantis™(Instem LSS Ltd., Staffordshire, England) computer software/database wasused for histopathology data acquisition, reporting and analysis.

Blood Collection and Processing

Blood collected at necropsy was processed to plasma by centrifugation ata minimum of 1300 g at 4° C. for 10 minutes. Plasma samples were storedat −70 to −90° C. until analysis.

Bioanalytical Analyses

Systemic blood (in the form of plasma from K₂EDTA) and lung tissue wasassayed via the liquid chromatography-mass spectrometry (LCMS) assay toquantify the amount of paclitaxel as a function of time. In brief theassay utilizes an ultra-performance liquid chromatography tandem massspectrometry (UPLC-MS/MS) assay to quantify paclitaxel. Samples areextracted via a protein precipitation method and separation is achievedvia reversed phase chromatography. Quantification was conducted with amatrix based calibration curve.

Non-compartmental analyses were conducted on data from the plasma andlung tissue concentrations. At a minimum the C_(max), T_(max), AUC andapparent terminal half-life were determined. Other parameters may bedetermined based on the data.

Results Clinical Observations, Survival, and Bodyweights

All animals survived to their designated necropsy timepoint. All animalswere euthanized within the window intended for each time point.

No abnormal clinical observations were noted through the duration of thestudy.

FIG. 51 and FIG. 52 show the average body weights through the durationof the study and as a percent change from Day 1. All groups gainedweight at about the same rate through the course of the study.

ABRAXANE® IV Tail Vein Injections

For the group receiving IV injections of ABRAXANE®, Day 1 bodyweightsranged from 386.1 to 472.8 g, this resulted in ABRAXANE® doses of2.6-3.2 mg/kg. The average dose (standard deviation) was 2.9 (0.16)mg/kg. Individual ABRAXANE® doses are shown in Table 9.

TABLE 9 Individual ABRAXANE ® Doses Day 1 ABRAXANE ® Subject Bodyweightadministered Dose Name (g) {circumflex over ( )}(mg) (mg/kg) 1001 442.11.25 2.8 1002 441.3 1.25 2.8 1003 425.I 1.25 2.9 1004 435.7 1.25 2.91005 446.3 1.25 2.8 1006 412.8 1.25 3.0 1007 472.8 1.25 2.6 1008 435.61.25 2.9 1009 400.4 1.25 3.1 1010 469.8 1.25 2.7 1011 412.9 1.25 3.01012 456.9 1.25 2.7 1013 390.7 1.25 3.2 1014 403.6 1.25 3.1 1015 414.11.25 3.0 1016 436.0 1.25 2.9 1017 404.5 1.25 3.1 1018 424.7 1.25 2.91019 386.1 1.25 3.2 1020 395.0 1.25 3.2 1021 414.8 1.25 3.0 1022 438.51.25 2.9 1023 458.7 1.25 2.7 1024 425.4 1.25 2.9 1025 467.3 1.25 2.71026 423.2 1.25 3.0 1027 414.8 1.25 3.0 1028 453.5 1.25 2.8 1029 441.11.25 2.8 1030 458.6 1.25 2.7 Average 430.1 1.3 2.9 Std. Dev. 24.14 0.000.16 {circumflex over ( )}Animals received a maximum IV dose volume of250 uL of the 5 mg/mL ABRAXANE ® formulation (1.25 mg).

Paclitaxel Particle Exposures

Aerosol Concentration and Particle Size

See: Results—Aerosol Concentration and Particle Size in Example 2.

Oxygen and Temperature

See: Results—Oxygen and Temperature in Example 2.

Deposited Dose

See: Results—Deposited Dose in Example 2. Necropsy

All animals survived to their designated necropsy timepoint. At necropsyanimals from each group had minimal to mild, tan discolorations on thelungs (Table 10). Such observations are often associated with inhalationexposures. Other sporadic observations included an enlarged heart(animal #2016) and enlarged tracheobronchial lymph nodes. No otherabnormal gross observations were noted at necropsy.

TABLE 10 Summary of Gross Necropsy Observations Low Dose High Dosepaclitaxel paclitaxel ABRAXANE ® particle particles Naive IV formulationIH Control Number on study 30 30 30 3 No visible lesions 15 14 11 3Lungs - Discoloration; Tan; All; Patchy Minimal (1) 0 4 2 0 Mild (2) 1412 15 0 Moderate (3) 1 0 2 0

Histopathology

There were no significant abnormalities noted within the trachea andleft lungs of the 336 hour (˜14 day) post-dosing sacrifice animalsexamined for this study. Tissues were microscopically indistinguishablefrom “Spare” animals serving as controls.

Macrophage accumulation was not apparent within the lung sections oftreatment animals examined for this study. Some level of increase inalveolar macrophages is very common in inhalation studies as aphysiologically normal response to exogenous material deposited in thelung (minor levels can also be a relatively common observation inuntreated animals). The apparent absence in inhalation dosed animals inthis study may be partly related to the relatively late (336 hour or ˜14day) post-dose timepoint examined histologically.

Bioanalytical and PK Modeling

Results are summarized below in Tables 11, 12, and 13, and in FIG. 53and FIG. 54. The average paclitaxel plasma concentration vs. time andaverage paclitaxel lung tissue concentration vs. time data was modeledas shown above and the results are shown in Table 14 and 15,respectively.

TABLE 11 Lung and Plasma Bioanalytical Results - ABRAXANE ® IV (IVnab-paclitaxel) Plasma Lung Tissue Mean Mean Concentration ConcentrationConcen- Per Concen- Per Animal Timepoint tration Timepoint trationTimepoint ID (hr) (ng/mL) (ng/mL) (ng/mL) (ng/mL) 1001 0.5 153 206 58505800 1002 205 5250 1003 261 6300 1004 6 70.5 62.2 2665 2730 1005 66.72880 1006 49.3 2645 1007 12 18.9 20.0 1045 1170 1008 20 1145 1009 21.11320 1010 24 9.46 15.3 386 647 1011 16.3 825 1012 20.1 730 1013 48 5.082.98 307 244 1014 1.56 190 1015 2.3 237 1016 72 BQL 1.05 101 145 10171.05 221 1018 BQL 113 1019 120 BQL BQL BQL BQL 1020 BQL BQL 1021 BQL BQL1022 168 BQL BQL BQL BQL 1023 BQL BQL 1024 BQL BQL 1025 240 BQL BQL BQLBQL 1026 BQL BQL 1027 BQL BQL 1028 336 BQL BQL BQL BQL 1029 BQL BQL 1030BQL BQL

TABLE 12 Lung and Plasma Bioanalytical Results - Paclitaxel ParticleFormulation Low Dose (0.38 mg/kg) IH Plasma Lung Tissue Mean MeanConcentration Concentration Concen- Per Concen- Per Animal Timepointtration Timepoint tration Timepoint ID (hr) (ng/mL) (ng/mL) (ng/mL)(ng/mL) 2001 0.5 15.6 11.6 19450 21000 2002 12.1 17700 2003 7.09 258502004 6 3.44 2.87 6700 4990 2005 2.37 3945 2006 2.81 4325 2007 12 5.293.35 6200 5368 2008 2.08 5550 2009 2.67 4355 2010 24 BQL 1.26 2325 30082011 1.16 2045 2012 1.36 4655 2013 48 BQL BQL 850 1247 2014 BQL 15302015 BQL 1360 2016 72 BQL BQL 950 950 2017 BQL 1385 2018 BQL 515 2019120 BQL BQL 1500 1045 2020 BQL 890 2021 BQL 745 2022 168 BQL BQL 309 3772023 BQL 695 2024 BQL 129 2025 240 BQL BQL 58 109 2026 BQL 151 2027 BQL117 2028 336 BQL BQL BQL 55.5 2029 BQL 55.5 2030 BQL BQL

TABLE 13 Lung and Plasma Bioanalytical Results - Paclitaxel ParticleFormulation High Dose (1.18 mg/kg) IH Plasma Lung Tissue Mean MeanConcentration Concentration Concen- Per Concen- Per Animal Timepointtration Timepoint tration Timepoint ID (hr) (ng/mL) (ng/mL) (ng/mL)(ng/mL) 3001 0.5 10.8 15.9 40400 41600 3002 21.3 43800 3003 15.6 406003004 6 6.56 5.69 15500 20800 3005 4.35 20400 3006 6.15 26500 3007 127.14 4.95 17050 14700 3008 3.47 13500 3009 4.23 13550 3010 24 1.47 1.9610300 11433 3011 3.11 11700 3012 1.31 12300 3013 48 1.21 1.21 6000 67003014 BQL 7300 3015 BQL 6800 3016 72 BQL 1.06 4375 3953 3017 1.06 47353018 BQL 2750 3019 120 BQL BQL 1570 1923 3020 BQL 1110 3021 BQL 30903022 168 BQL BQL 3395 2143 3023 BQL 1410 3024 BQL 1625 3025 240 BQL BQL271 430 3026 BQL 448 3027 BQL 570 3028 336 BQL BQL 233 272 3029 BQL 3673030 BQL 216

TABLE 14 Paclitaxel plasma PK modeling results AUC_(D(last)) DoseC_(max) T_(max) T_(1/2) AUC_((last)) (hr*ng*mg/ Group (mg/kg) (ng/mL)(hr) (hr) (hr*ng/mL) mL*kg) IV 2.9 206 0.5 8.7 1517 528 Inha- 0.38 11.60.5 7.9 101 264 lation Inha- 1.18 15.9 0.5 8.6 228 193 lation

TABLE 15 Paclitaxel lung tissue PK modeling results AUC_(D(last)) DoseC_(max) T_(max) T_(1/2) AUC_((last)) (hr*ng*mg/ Group (mg/kg) (ng/mL)(hr) (hr) (hr*ng/mL) mL*kg) IV 2.9 5800 0.5 19.9 62,870 23,112 Inha-0.38 21,000 0.5 56.3 342,877 914,095 lation Inha- 1.18 41,600 0.5 56.01,155,662 997,985 lation

The modeling was conducted with WinNonlin based on average plasma orlung tissue concentrations at each time point. The NCA was designed toquantify the exposure (area under the concentration versus time curve[AUC]), time to maximum concentration (T_(max)), maximum concentration(C_(max)) and when possible apparent terminal half-life (T_(1/2)). Thehalf-life within systemic plasma was unchanged for the formulation/dosestested and the half-life within the lung tissue was increased with thepaclitaxel particle formulation delivered by inhalation. The exposure tothe lung tissue (dose normalized AUC) was increased when delivered asthe paclitaxel particle formulation by inhalation.

Collectively the data indicate a significant retention of paclitaxelparticles within the lung tissue when delivered via inhalation.

Conclusions

Ninety (90) male Sprague Dawley rats were exposed to “clinicalreference” dose of paclitaxel, ABRAXANE® (paclitaxel protein boundparticles for injectable suspension), by intravenous (IV) bolusinjection or paclitaxel particle formulation (target dose of 0.37 or 1.0mg/kg) by nose only inhalation on a single occasion. Three animals (n=3)were euthanatized at ten (10) timepoints from 0.5 to 336 hours postexposure for blood (plasma) and lung tissue collections.Non-compartmental analysis was performed on plasma and lung tissue toidentify the duration of detectable amounts of paclitaxel post exposurefor each dose group. Animals designated to the 336 hour time point fromall groups had right lungs collected for liquid chromatography-massspectrometry (LCMS) analysis while the left lungs were perfused with 10%neutral buffered formalin (NBF) and retained for potentialhistopathology. In order to enable comparative histopathology, threespare animals (Native Controls) were also euthanized at the 336 hourtimepoint and had lung collections performed in the same manner. Animalsdesignated to all other timepoints had all lungs individually frozen forLCMS analysis.

The inhalation exposure average Paclitaxel aerosol concentration for LowDose and High Dose paclitaxel particle formulation groups was of 85.64μg/L and 262.27 μg/L, respectively. The average exposure aerosolconcentration was within ±15% of target aerosol concentration which wasexpected for nebulized inhalation exposures. The particle sizedistribution was determined in terms of MMAD (GSD) for each paclitaxelparticle formulation aerosols using cascade impactor. For 6.0 mg/mL and20.0 mg/mL paclitaxel particle formulation aerosols the MMAD (GSD) weredetermined to be 1.8 (2.0) μm and 2.3 (1.9) μm, respectively.

Paclitaxel deposited dose was calculated based on Paclitaxel averageaerosol concentration of 85.64 μg/L, average Day 0 group bodyweight of420.4 g, assumed deposition fraction of 10% and exposure duration of 65minutes; the average achieved rodent deposited dose was determined to be0.38 mg/kg for the Low Dose paclitaxel particle formulation group. Forthe High Dose paclitaxel particle formulation group, paclitaxel averageaerosol concentration of 262.27 μg/L, average Day 0 group bodyweight of420.5 g, assumed deposition fraction of 10% and exposure duration of 65minutes; the average achieved rodent deposited dose was determined to be1.18 mg/kg. The recorded oxygen and temperature ranges were 19.8%-20.9%and 20.7° C.-20.8° C., respectively for 6.0 mg/mL paclitaxel particleformulation exposure. For 20.0 mg/mL paclitaxel particle formulationexposure, the recorded oxygen value was 19.8% throughout the exposureand temperature range was 20.7° C.-20.8° C.

For the group receiving IV injections of ABRAXANE®, Day 1 bodyweightsranged from 386.1 to 472.8 g, this resulted in ABRAXANE® doses of2.6-3.2 mg/kg, with the average group dose being 2.9 mg/kg.

All groups gained weight through the course of the study. No abnormalclinical observations were noted through the duration of the study. Allanimals survived to their designated necropsy timepoint. All animalswere euthanized within the window intended for each time point.

At necropsy, approximately half of the animals from each group hadminimal to mild, tan discolorations on the lungs. Such observations areoften associated with inhalation exposures. Other transient observationsincluded an enlarged heart (animal #2016) and enlarged tracheobronchiallymph nodes. No other abnormal gross observations were noted atnecropsy. Histopathology showed lung and trachea from test and referencearticle treated rats were within normal limits and indistinguishablefrom those of naive rats under the conditions of this study.

The NCA was designed to quantify the exposure (area under theconcentration versus time curve [AUC]), time to maximum concentration(T_(max)), maximum concentration (C_(max)) and when possible apparentterminal half-life (T_(1/2)).

The hypothesis for the novel paclitaxel particle formulation was thatthe formulation would result in increased retention of paclitaxel withinthe lung tissue and reduce the systemic exposure. The half-life withinsystemic plasma was unchanged for the formulation/doses tested and thehalf-life within the lung tissue was increased with the paclitaxelparticle formulation delivered by inhalation. The exposure to the lungtissue (dose normalized AUC) was increased when delivered as thepaclitaxel particle formulation by inhalation.

Collectively the data indicate a significant retention of paclitaxelparticles within the lung tissue when delivered via inhalation comparedto the IV “clinical reference”.

We claim:
 1. A method for treating a lung disorder, including but notlimited to a lung tumor or pulmonary fibrosis, comprising pulmonaryadministration to a subject with a lung disorder of an amount effectiveof a composition comprising taxane particles to treat the lung disorder,wherein the taxane particles comprise at least 95% of the taxane andhave a mean particle size (number) of between 0.1 μm and 5 μm.
 2. Themethod of claim 1, wherein the pulmonary administration comprisesnebulization, and wherein the nebulization results in pulmonary deliveryto the subject of aerosol droplets of the taxane particles or suspensionthereof.
 3. The method of claim 1, wherein the taxane particles have amean particle size (number) of between 0.4 μm and 2 μm.
 4. The method ofclaim 1, wherein the taxane particles have a mean particle size (number)of between about 0.4 μm and about 1.2 μm.
 5. The method of claim 1,wherein the taxane particles have a specific surface area (SSA) of atleast 10 m²/g, or at least 12 m²/g, 14 m²/g, 16 m²/g, 18 m²/g, 20 m²/g,25 m²/g, 30 m²/g, 32 m²/g, 34 m²/g, or 35 m²/g.
 6. The method of claim1, wherein the taxane particles are present in a suspension, wherein thesuspension comprises: (a) the taxane particles; (b) a pharmaceuticallyacceptable carrier; and (c) a polysorbate, wherein the polysorbate ispresent in the suspension at a concentration of between about 0.01% v/vand about 1.5% v/v.
 7. The method of claim 6, wherein thepharmaceutically acceptable carrier is saline.
 8. The method of claim 6,wherein the polysorbate is polysorbate
 80. 9. The method of claim 6,wherein the taxane is present in the suspension at a concentrationbetween about 1 mg/ml and about 40 mg/ml.
 10. The method of claim 1,wherein the particles and suspensions thereof are uncoated and excludelipids, polymers, proteins such as albumin, polyethoxylated castor oil,and/or polyethylene glycol glycerides composed of mono-, di- andtriglycerides and mono- and diesters of polyethylene glycol.
 11. Themethod of claim 1, wherein the taxane comprises paclitaxel, docetaxel,cabazitaxel, or a pharmaceutically acceptable salt thereof.
 12. Themethod of claim 11, wherein the taxane comprises paclitaxel or apharmaceutically acceptable salt thereof.
 13. The method of claim 12,wherein the particles have one or more of the following characteristics:(a) a mean bulk density (not tapped) between about 0.050 g/cm³ and about0.12 g/cm³; (b) a SSA of at least 12 m²/g; (c) a SSA of between about 22m²/g and about 40 m²/g; and/or (d) wherein at least 40% (w/w) of thepaclitaxel is dissolved in 30 minutes or less in a solution of 50%methanol/50% water (v/v) at 37° C. and pH 7.0 in a USP II paddleapparatus operating at 75 RPM.
 14. The method of claim 11, wherein thetaxane comprises docetaxel or a pharmaceutically acceptable saltthereof.
 15. The method of claim 14, wherein the particles have one ormore of the following characteristics: (a) a mean bulk density (nottapped) between about 0.050 g/cm³ and about 0.12 g/cm³; (b) a SSA of atleast 12 m²/g; (c) a SSA of between about 20 m²/g and about 50 m²/g;and/or (d) wherein at least 20% (w/w) of the docetaxel is dissolved in30 minutes or less in a solution of 15% methanol/85% water (v/v) at 37°C. and pH 7.0 in a USP II paddle apparatus operating at 75 RPM.
 16. Themethod of claim 1, wherein the taxane remains detectable in lung tissueof the subject for at least 4 days after the administering.
 17. Themethod of claim 1, wherein the taxane particles are in crystalline form.18. The method of claim 1, wherein the taxane particles or suspensionsthereof are aerosolized for administration, and aerosol dropletsresulting from aerosolization have a mass median aerodynamic diameter(MMAD) of between about 0.5 μm to about 6 μm diameter.
 19. The method ofclaim 1, wherein the lung disorder comprises a lung tumor, and whereinthe taxane particles reside at the lung tumor site after administrationof the composition exposing the tumor to the taxane particles for asustained amount of time sufficient to stimulate the subject'sendogenous immune system resulting in production of tumoricidal cellsand infiltration of the tumoricidal cells into the lung tumor at a levelsufficient to treat the tumor.
 20. The method of claim 19, wherein thesustained amount of time is at east 4 weeks.
 21. The method of claim 19,wherein the tumoricidal cells comprise T-cells, B cells, or naturalkiller (NK) cells, or combinations thereof.
 22. The method of claim 1,wherein the composition is administered in two or more separateadministrations.
 23. The method of claim 22, wherein the two or moreseparate administrations are administered once a week for at least twoweeks.
 24. The method of claim 22, wherein the two or more separateadministrations are administered twice a week for at least one week,wherein the two or more separate administrations are separated by atleast one day.
 25. The method of claim 1, wherein the lung disordercomprises a lung tumor, and wherein the treating comprises eliminationof the tumor.